DNA molecules encoding Ctenocephalides felis glutamate gated chloride channels

ABSTRACT

To date, L-glutamate-gated chloride (GluCl) channels have been observed only in invertebrate organisms. Modulators of this channel (either agonists or antagoinists) will interfere with neurotransmission. For example, agents such as avermectins activate the GluCl, causing paralysis due to blocking of neurotranmitter release, resulting in death of the organism. Because GluCl channels are invertebreate specific, they are excellent targets for the discovery of novel insecticides, anthelminths and parasiticides that will display a marked safety profile because of the lack of mechanism based toxicity in vertebrate organisms. The present specification discloses isolation of a cDNA clone from the cat flea  Ctenocephalides felis  (CfGluCl-1) that encodes a L-glutamate-gated chloride channel. Heterologous expression of CfGluCl-1 cRNA in  Xenopus oocytes  results in robust expression of a L-glutamate-gated chloride current and the channel is activated and potentiated by avermectins. The expression of CfGluCl-1 in a heterologous expression system if useful to screens for novel GluCl channel agonsts and antagonsits. Additionally, this specification disclose impoved methods of screening for GluCl channel modulators.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of provisional application number 60/055,451filed Aug. 11, 1997.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not applicable.

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

FIELD OF THE INVENTION

The present invention relates in part to isolated nucleic acid molecules(polynucleotides) which encode Ctenocephalides felis (flea) glutamategated chloride channels. The present invention also relates torecombinant vectors and recombinant hosts which contain a DNA fragmentencoding C. felis glutamate gated chloride channels, substantiallypurified forms of associated C. felis glutamate gated chloride channels,associated mutant proteins, and methods associated with identifyingcompounds which modulate associated Ctenocephalides felis glutamategated chloride channels, which will be useful as insecticides.

BACKGROUND OF THE INVENTION

Glutamate-gated chloride channels, or H-receptors, have been identifiedin arthropod nerve and muscle (Lingle et al, 1981, Brain Res. 212:481-488; Horseman et al., 1988, Neurosci. Lett. 85: 65-70; Wafford andSattelle, 1989, J. Exp. Bio. 144:449-462; Lea and Usherwood, 1973, Comp.Gen. Parmacol. 4: 333-350; and Cull-Candy, 1976, J. Physiol.255:449-464).

Additionally, glutamate-gated chloride channels have been cloned fromthe soil nematode Caenorhabditis elegans (Cully et al., 1994, Nature371: 707-711; see also U.S. Pat. No. 5,527,703) and Drosophilamelanogaster (Cully et al., 1996, J. Biol. Chem. 271: 20187-20191).

Invertebrate glutamate-gated chloride channels are important targets forthe widely used avermectin class of anthelmintic and insecticidalcompounds. The avermectins are a family of macrocyclic lactonesoriginally isolated from the actinomycete Streptomyces avermitilis. Thesemisynthetic avermectin derivative, ivermectin(22,23-dihydro-avermectin B_(1a)), is used throughout the world to treatparasitic helminths and insect pests of man and animals. The avermectinsremain the most potent broad spectrum endectocides exhibiting lowtoxicity to the host. After many years of use in the field, thereremains little resistance to avermectin in the insect population. Thecombination of good therapeutic index and low resistance stronglysuggests that the glutamate-gated chloride (GluCl) channels remain goodtargets for insecticide development.

It would be advantageous to identify additional invertebrate genesencoding encoding GluCl channels in order to allow screening to identifynovel GluCl channel modulators that may have insecticidal, mitacidaland/or nematocidal activity for animal health or crop protection. Thepresent invention addresses and meets these needs by disclosing isolatednucleic acid molecules which express a Ctenocephalides felis GluGlchannel wherein expression of flea GluCl cRNA in Xenopus oocytes resultsin an active GluCl channel.

SUMMARY OF THE INVENTION

The present invention relates to isolated nucleic acid molecules(polynucleotides) which encode novel invertebrate GluCl channelproteins, especially nucleic acid molecules which encode a functional C.felis GluCl (herein, “CfGluCl”) channel.

The present invention also relates to isolated nucleic acid fragments ofCfGluCl which encode mRNA expressing a biologically active CfGluClchannel. Any such polynucleotide includes but is not necessarily limitedto nucleotide substitutions, deletions, additions, amino-terminaltruncations and carboxy-terminal truncations such that these mutationsencode cRNA which express a functional C. felis GluCl channel in aeukaryotic cell, such as Xenopus oocytes, so as to be useful forscreening for agonists and/or antagonists of C. felis GluCl activity.

The isolated nucleic acid molecule of the present invention may includea deoxyribonucleic acid molecule (DNA), such as genomic DNA andcomplementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA), including but not limited to messenger RNA (mRNA)encoding a biologically active C. felis GluCl channel and complementaryRNA (cRNA) transcribed from a recombinant expression vector comprising aDNA molecule which encodes a full-length or biologically active portionof the full-length C. felis GluCl channel.

A preferred aspect of the present invention is disclosed in FIGS. 1A-Band SEQ ID NO:1, an isolated cDNA molecule encoding a C. felis GluClchannel, CfGluCl-1.

The present invention relates to recombinant vectors and recombinanthosts, both prokaryotic and eukaryotic, which contain the substantiallypurified nucleic acid molecules disclosed throughout this specification,especially a nucleic acid molecule encoding a C. felis GluCl channel,CfGluCl, such as the cDNA molecule disclosed in FIGS. 1A-B and set forthin SEQ ID NO:1.

The present invention also relates to a substantially purified form of aC. felis GluCl channel protein and especially the C. felis GluCl channeldisclosed in FIG. 2 and set forth in SEQ ID NO:2.

The present invention relates to a substantially purified membranepreparation which comprises a C. felis GluCl channel and is essentiallyfree from contaminating proteins, including but not limited to other C.felis source proteins or host proteins from a recombinant cell whichexpresses CfGluCl. Especially preferred is a membrane preparation whichcomprises C. felis GluCl channel disclosed in FIG. 2 and set forth inSEQ ID NO:2. To this end, the present invention also relates to asubstantially purified membrane preparation which is purified from arecombinant host, whether a recombinant eukaryotic or recombinantprokaryotic host, wherein a recombinant vector expresses a C. felisGluCl channel. Especially preferred is a membrane preparation whichcomprises a recombinant form of the C. felis GluCl channel, CfGluCl,disclosed in FIG. 2 and set forth in SEQ ID NO:2, referred to asCfGluCl-1.

The present invention also relates to biologically active fragmentsand/or mutants of a C. felis GluCl channel protein, including but notlimited to the CfGluCl protein disclosed in FIG. 2 and set forth in SEQID NO:2, including but not necessarily limited to amino acidsubstitutions, deletions, additions, amino terminal truncations andcarboxy-terminal truncations such that these mutations provide for abiologically active channel which is useful in screening for agonistsand/or antagonists of C. felis GluCl channel activity.

The present invention also relates to an isolated nucleic acid molecule(polynucleotide) which encodes a truncated form of the flea GluClchannel protein (herein, “tr-CfGluCl”), as exemplified in FIG. 3 and setforth in SEQ ID NO:3. Co-expression of tr-CfGluCl in Xenopus oocyteswith CfGluCl is shown to inhibit glutamate-gated channel activity.

The present invention also relates to isolated nucleic acid fragments oftr-CfGluCl-1 (SEQ ID NO:3) which encodes cRNA expressing a biologicallyactive form of tr-CfGluCl, including but not limited to inhibition orpromotion of CfGluCl channel activity in the target cell type. Any suchpolynucleotide includes but is not necessarily limited to nucleotidesubstitutions, deletions, additions, amino-terminal truncations andcarboxy-terminal truncations from the truncated form.

Again, any such truncated nucleic acid molecule (as compared to CfGluCl)may include a deoxyribonucleic acid molecule (DNA), such as genomic DNAand complementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA), including but not limited to messenger RNA (mRNA) orcomplementary RNA (cRNA) transcribed from a recombinant expressionvector comprising a DNA molecule which encodes a truncated version ofthe full-length C. felis GluCl channel.

A preferred aspect of this portion of the invention is disclosed inFIGS. 3A-B and SEQ ID NO:4, an isolated cDNA molecule encoding atruncated version of the C. felis GluCl channel.

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which contain thesubstantially purified nucleic acid molecules disclosed throughout thisspecification, especially a nucleic acid molecule encoding a truncatedversion of a C. felis GluCl channel such as the cDNA molecule disclosedin FIGS. 3A-B and set forth in SEQ ID NO:3.

The present invention also relates to a substantially purified form of atruncated version of the C. felis GluCl channel, trCfGluCl, andespecially the truncated version of the C. felis GluCl channel, which isdisclosed in FIG. 4 and as set forth in SEQ ID NO:4, referred to astrCfGluCl-1.

The present invention also relates to biologically active fragmentsand/or mutants of the truncated C. felis GluCl channel, trCfGluCl-1,including but not necessarily limited to amino acid substitutions,deletions, additions, amino terminal truncations and carboxy-terminaltruncations.

It is an object of the present invention to provide an isolated nucleicacid molecule which encodes a novel form of a C. felis GluCl channel andbiologically active fragments thereof which are derivatives of SEQ IDNO:2.

It is a further object of the present invention to provide the C. felisGluCl channel proteins or protein fragments encoded by the nucleic acidmolecules referred to in the preceding paragraph.

It is a further object of the present invention to provide recombinantvectors and recombinant host cells which comprise a nucleic acidsequence encoding a C. felis GluCl channel or a biological equivalentthereof.

It is an object of the present invention to provide a substantiallypurified form of a C. felis GluCl channel or a biological equivalentthereof, as set forth in SEQ ID NO:2.

It is also an object of the present invention to provide a membranepreparation membrane preparation which comprises a C. felis GluClchannel and is essentially free from contaminating proteins. Thismembrane preparation includes, but is not limited to, a membranepreparation purified from a recombinant host.

It is an object of the present invention to provide for biologicallyactive fragments and/or mutants of CfGluCl, including but notnecessarily limited to amino acid substitutions, deletions, additions,amino terminal truncations and carboxy-terminal truncations such thatthese mutations provide for proteins or protein fragments of diagnostic,therapeutic or prophylactic use.

It is an object of the present invention to provide a substantiallypurified form of CfGluCl-1, as set forth in SEQ ID NO:4.

It is an object of the present invention to provide for biologicallyactive fragments and/or mutants of CfGluCl, including but notnecessarily limited to amino acid substitutions, deletions, additions,amino terminal truncations and carboxy-terminal truncations.

As used herein, “GluCl” refers to a glutamate-gated chloride channel.

As used herein, “CfGluCl” refers to a biologically active form of a C.felis glutamate-gated chloride channel.

As used herein, “cDNA” refers to complementary DNA.

As used herein, “mRNA” refers to messenger RNA.

As used herein, “cRNA” refers to complementary RNA, transcribed from arecombinant cDNA template.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B shows the nucleotide sequence which comprises the openreading frame encoding the C. felis GluCl channel, CfGluCl-1 (SEQ IDNO:1).

FIG. 2 shows the amino acid sequence of CfGluCl-1 (SEQ ID NO:2).

FIGS. 3A-B shows the nucleotide sequence which comprises the openreading frame encoding the truncated C. felis GluCl channel, trCfGluCl-1(SEQ ID NO:3).

FIG. 4 shows the amino acid sequence of trCfGluCl-1 (SEQ ID NO:4).

FIGS. 5A and 5B show activation of CfGluCl-1 by glutamate. FIG. 5A showssuperimposed current recordings in response to 10, 30, 100 and 300 μMglutamate. FIG. 5B shows the concentration-response curve for glutamate.

FIG. 6 shows that the CfGluCl-1 channel is selective for chloride.

FIGS. 7A and 7B show that ivermectin phosphate (IVM-PO₄) is an agonistof the C. felis GluCl channel encoded by CfGluCl-1. FIG. 7A showssuperimposed current recordings showing activation by 100 μM glutamateand 10 nM IVM-PO₄. FIG. 7B shows the concentration-response curve forIVM-PO₄ for CfGluCl (0 mV), DmGluCl (0 mV) and DmGluCl (−80 mV).

DETAILED DESCRIPTION OF THE INVENTION

L-glutamate-gated chloride (GluCl) channels have been observed only ininvertebrate organisms. A modulator of this channel (either an agonistor antagonist) will interfere with neurotransmission. Agents such asavermectins activate this channel and cause paralysis due to block ofneurotranmitter release, resulting in death of the organism. BecauseGluCl channels are invertebrate specific, they are excellent targets forthe discovery of novel insecticides, anthelminthics and parasiticidesthat will display a marked safety profile because of the lack ofmechanism based toxicity in vertebrate organisms. The present inventionrelates to isolated nucleic acid molecules (polynucleotides) whichencode novel invertebrate GluCl channel proteins, especially nucleicacid molecules which encode a functional C. felis GluCl channel (herein,“CfGluCl”). Heterologous expression of CfGluCl cRNA in Xenopus oocytesresults in robust expression of a L-glutamate-gated chloride current.The CfGluCl channel is activated and potentiated by avermectins (e.g.,ivermectin phosphate). The expression of CfGluCl-1 in a heterologousexpression system can be used to establish screens for novel GluClchannel modulators. Such compounds will be useful as antiparasitics andinsecticides in human and animal health and crop protection, becausethey will be devoid of mechanism based vertebrate toxicity.

To this end, the present invention also relates to isolated nucleic acidfragments of CfGluCl which encode cRNA expressing a biologically CfGluClchannel. Any such polynucleotide includes but is not necessarily limitedto nucleotide substitutions, deletions, additions, amino-terminaltruncations and carboxy-terminal truncations such that these mutationsencode cRNA which express a functional C. felis GluCl channel in aeukaryotic cell, such as Xenopus oocytes, so as to be useful forscreening for agonists and/or antagonists of C. felis GluCl activity.

A preferred aspect of the present invention is disclosed in FIGS. 1A-Band SEQ ID NO:1, an isolated cDNA molecule encoding a C. felis GluClchannel, CfGluCl-1.

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which contain thesubstantially purified nucleic acid molecules disclosed throughout thisspecification, especially a nucleic acid molecule encoding a C. felisGluCl channel, CfGluCl, such as the cDNA molecule disclosed in FIGS.1A-B and set forth in SEQ ID NO:1.

The isolated nucleic acid molecule of the present invention may includea deoxyribonucleic acid molecule (DNA), such as genomic DNA andcomplementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA), including but not limited to messenger RNA (mRNA)encoding a biologically active C. felis GluCl channel and complementaryRNA (cRNA) transcribed from a recombinant expression vector comprising aDNA molecule which encodes a full-length or biologically active portionsof the full-length C. felis GluCl channel.

It is known that there is a substantial amount of redundancy in thevarious codons which code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences transcribing mRNA orcRNA comprising alternative codons which encode an identical amino acid,as shown below:

A=Ala=Alanine: codons GCA, GCC, GCG, GCU

C=Cys=Cysteine: codons UGC, UGU

D=Asp=Aspartic acid: codons GAC, GAU

E=Glu=Glutamic acid: codons GAA, GAG

F=Phe=Phenylalanine: codons UUC, ULTUU

G=Gly=Glycine: codons GGA, GGC, GGG, GGU

H=His=Histidine: codons CAC, CAU

I=Ele=Isoleucine: codons AUA, AUC, AUU

K=Lys=Lysine: codons AAA, AAG

L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU

M=Met=Methionine: codon AUG

N=Asp=Asparagine: codons AAC, AAU

P=Pro=Proline: codons CCA, CCC, CCG, CCU

Q=Gln=Glutamine: codons CAA, CAG

R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU

S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU

T=Thr=Threonine: codons ACA, ACC, ACG, ACU

V=Val=Valine: codons GUA, GUC, GUG, GUU

W=Trp=Tryptophan: codon UGG

Y=Tyr=Tyrosine: codons UAC, UAU

Therefore, the present invention discloses codon redundancy which mayresult in differing DNA molecules expressing an identical protein. Forpurposes of this specification, a sequence bearing one or more replacedcodons will be defined as a degenerate variation. Also included withinthe scope of this invention are mutations either in the DNA sequence orthe translated protein which do not substantially alter the ultimatephysical properties of the expressed protein. For example, substitutionof valine for leucine, arginine for lysine, or asparagine for glutaminemay not cause a change in functionality of the polypeptide.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude but are not limited to site-directed mutagenesis. Examples ofaltered properties include but are not limited to changes in theaffinity of an enzyme for a substrate or a receptor for a ligand.

As used herein, “purified” and “isolated” are utilized interchangeablyto stand for the proposition that the nucleic acid, protein, orrespective fragment thereof in question has been substantially removedfrom its in vivo environment so that it may be manipulated by theskilled artisan, such as but not limited to nucleotide sequencing,restriction digestion, site-directed mutagenesis, and subcloning intoexpression vectors for a nucleic acid fragment as well as obtaining theprotein or protein fragment in pure quantities so as to afford theopportunity to generate polyclonal antibodies, monoclonal antibodies,amino acid sequencing, and peptide digestion. Therefore, the nucleicacids claimed herein may be present in whole cells or in cell lysates orin a partially purified or substantially purified form. A nucleic acidis considered substantially purified when it is purified away fromenvironmental contaminants. Thus, a nucleic acid sequence isolated fromcells is considered to be substantially purified when purified fromcellular components by standard methods while a chemically synthesizednucleic acid sequence is considered to be substantially purified whenpurified from its chemical precursors.

The present invention also relates to a substantially purified form of aC. felis GluCl channel, CfGluCl, and especially the C. felis GluClchannel which is disclosed in FIG. 2 and as set forth in SEQ ID NO:2,referred to as CfGluCl-1.

The present invention also relates to a substantially purified membranepreparation which comprises a C. felis GluCl channel and is essentiallyfree from contaminating proteins. Especially preferred is a membranepreparation which comprises a C. felis GluCl channel disclosed in FIG. 2and set forth in SEQ ID NO:2, referred to as CfGluCl-1.

The present invention also relates to a substantially purified membranepreparation which is purified from a recombinant host, whether arecombinant eukaryotic or recombinant prokaryotic host, wherein arecombinant vector expresses a C. felis GluCl channel. Especiallypreferred is a membrane preparation which comprises a recombinant formof the C. felis GluCl channel, CfGluCl, disclosed in FIG. 2 and setforth in SEQ ID NO:2, referred to as CfGluCl-1.

The present invention also relates to biologically active fragmentsand/or mutants of CfGluCl-1, including but not necessarily limited toamino acid substitutions, deletions, additions, amino terminaltruncations and carboxy-terminal truncations such that these mutationsprovide for a biologically active channel which is useful in screeningfor agonists and/or antagonists of C. felis GluCl channel activity.

As used herein, a “biologically active equivalent” or “functionalderivative” of a wild-type C. felis GluCl channel possesses a biologicalactivity that is substantially similar to the biological activity of thewild type C. felis GluCl channel. The term “functional derivative” isintended to include the “fragments,” “mutants,” “variants,” “degeneratevariants,” “analogs” and “homologues” or to “chemical derivatives” ofthe wild type C. felis GluCl channel protein. The term “fragment” ismeant to refer to any polypeptide subset of a wild-type C. felis GluClchannel. The term “mutant” is meant to refer to a molecule that may besubstantially similar to the wild-type form but possesses distinguishingbiological characteristics. Such altered characteristics include but arein no way limited to altered substrate binding, altered substrateaffinity and altered sensitivity to chemical compounds affectingbiological activity of the C. felis GluCl channel and/or C. felis GluClchannel derivative. The term “variant” is meant to refer to a moleculesubstantially similar in structure and function to either the entirewild-type protein or to a fragment thereof A molecule is “substantiallysimilar” to a wild-type C. felis GluCl channel and/or C. felis GluClchannel-like protein if both molecules have substantially similarstructures or if both molecules possess similar biological activity.Therefore, if the two molecules possess substantially similar activity,they are considered to be variants even if the structure of one of themolecules is not found in the other or even if the two amino acidsequences are not identical. The term “analog” refers to a moleculesubstantially similar in function to either the full-length C. felisGluCl channel and/or C. felis GluCl channel or to a biologically activefragment thereof.

The present invention also relates to isolated an isolated nucleic acidmolecule (polynucleotide) which encodes a truncated form of the fleaGluCl channel protein (herein, “tr-CfGluCl”), as exemplified in FIGS.3A-B and SEQ ID NO:3. Co-expression of tr-CfGluCl in Xenopus oocyteswith CfGluCl inhibits glutamate-gated channel activity.

The present invention also relates to isolated nucleic acid fragments ofSEQ ID NO:3 which encode cRNA expressing a biologically active form oftr-CfGluCl, including but not limited to inhibition or promotion ofCfGluCl channel activity in the target cell type. Any suchpolynucleotide includes but is not necessarily limited to nucleotidesubstitutions, deletions, additions, amino-terminal truncations andcarboxy-terminal truncations from the truncated form.

Again, any such truncated nucleic acid molecule (as compared to CfGluCl)may include a deoxyribonucleic acid molecule (DNA), such as genomic DNAand complementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA), including but not limited to messenger RNA (mRNA) orcomplementary RNA (cRNA) transcribed from a recombinant expressionvector comprising a DNA molecule which encodes a truncated version ofthe full-length C. felis GluCl channel.

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which contain thesubstantially purified nucleic acid molecules disclosed throughout thisspecification, especially a nucleic acid molecule encoding a truncatedversion of a C. felis GluCl channel, CfGluCl., such as the cDNA moleculedisclosed in FIGS. 3A-B and set forth in SEQ ID NO:3.

The present invention also relates to a substantially purified form of atruncated version of the C. felis GluCl channel, trCfGluCl, andespecially the truncated version of theC. felis GluCl channel, which isdisclosed in FIG. 4 and as set forth in SEQ ID NO:4, referred to astrCfGluCl-1.

The present invention also relates to biologically active fragmentsand/or mutants of trCfGluCl-1, including but not necessarily limited toamino acid substitutions, deletions, additions, amino terminaltruncations and carboxy-terminal truncations.

Any of a variety of procedures may be used to clone a C. felis GluClchannel. These methods include, but are not limited to, (1) a RACE PCRcloning technique (Frohman, et al., 1988, Proc. Natl. Acad. Sci. USA 85:8998-9002). 5′ and/or 3′ RACE may be performed to generate a full-lengthcDNA sequence. This strategy involves using gene-specificoligonucleotide primers for PCR amplification of C. felis GluCl channelcDNA. These gene-specific primers are designed through identification ofan expressed sequence tag (EST) nucleotide sequence which has beenidentified by searching any number of publicly available nucleic acidand protein databases; (2) direct functional expression of the C. felisGluCl channel cDNA following the construction of a C. felis GluClchannel-containing cDNA library in an appropriate expression vectorsystem; (3) screening a C. felis GluCl channel-containing cDNA libraryconstructed in a bacteriophage or plasmid shuttle vector with a labeleddegenerate oligonucleotide probe designed from the amino acid sequenceof the C. felis GluCl channel protein; and (4) screening a C. felisGluCl channel-containing cDNA library constructed in a bacteriophage orplasmid shuttle vector with a partial cDNA encoding the C. felis GluClchannel protein. This partial EDNA is obtained by the specific PCRamplification of C. felis GluCl channel DNA fragments through the designof degenerate oligonucleotide primers from the amino acid sequence knownfor other kinases which are related to the C. felis GluCl channelprotein; (5) screening a C. felis GluCl channel-containing cDNA libraryconstructed in a bacteriophage or plasmid shuttle vector with a partialcDNA encoding the C. felis GluCl channel protein. This strategy may alsoinvolve using gene-specific oligonucleotide primers for PCRamplification of C. felis GluCl channel cDNA identified as an EST asdescribed above; or (6) designing 5′ and 3′ gene specificoligonucleotides using SEQ ID NO: 1 as a template so that either thefull-length cDNA may be generated by known RACE techniques, or a portionof the coding region may be generated by these same known RACEtechniques to generate and isolate a portion of the coding region to useas a probe to screen one of numerous types of cDNA and/or genomiclibraries in order to isolate a full-length version of the nucleotidesequence encoding C. felis GluCl channel.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have C. felisGluCl channel activity. The selection of cells or cell lines for use inpreparing a cDNA library to isolate a cDNA encoding C. felis GluClchannel may be done by first measuring cell-associated C. felis GluClchannel activity using any known assay available for such a purpose.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Complementary DNA libraries may also be obtained from numerouscommercial sources, including but not limited to Clontech Laboratories,Inc. and Stratagene.

It is also readily apparent to those skilled in the art that DNAencoding C. felis GluCl channel may also be isolated from a suitablegenomic DNA library. Construction of genomic DNA libraries can beperformed by standard techniques well known in the art. Well knowngenomic DNA library construction techniques can be found in Sambrook, etal., supra.

In order to clone the C. felis GluCl channel gene by one of thepreferred methods, the amino acid sequence or DNA sequence of C. felisGluCl channel or a homologous protein may be necessary. To accomplishthis, the C. felis GluCl channel protein or a homologous protein may bepurified and partial amino acid sequence determined by automatedsequenators. It is not necessary to determine the entire amino acidsequence, but the linear sequence of two regions of 6 to 8 amino acidscan be determined for the PCR amplification of a partial C. felis GluClchannel DNA fragment. Once suitable amino acid sequences have beenidentified, the DNA sequences capable of encoding them are synthesized.Because the genetic code is degenerate, more than one codon may be usedto encode a particular amino acid, and therefore, the amino acidsequence can be encoded by any of a set of similar DNA oligonucleotides.Only one member of the set will be identical to the C. felis GluClchannel sequence but others in the set will be capable of hybridizing toC. felis GluCl channel DNA even in the presence of DNA oligonucleotideswith mismatches. The mismatched DNA oligonucleotides may stillsufficiently hybridize to the C. felis GluCl channel DNA to permitidentification and isolation of C. felis GluCl channel encoding DNA.Alternatively, the nucleotide sequence of a region of an expressedsequence may be identified by searching one or more available genomicdatabases. Gene-specific primers may be used to perform PCRamplification of a cDNA of interest from either a cDNA library or apopulation of cDNAs. As noted above, the appropriate nucleotide sequencefor use in a PCR-based method may be obtained from SEQ ID NO: 1, eitherfor the purpose of isolating overlapping 5′ and 3′ RACE products forgeneration of a full-length sequence coding for C. felis GluCl channel,or to isolate a portion of the nucleotide sequence coding for C. felisGluCl channel for use as a probe to screen one or more cDNA- orgenomic-based libraries to isolate a full-length sequence encoding C.felis GluCl channel or C. felis GluCl channel-like proteins.

In an exemplified method, a C. felis GluCl channel cDNA was generated byscreening a C. felis cDNA library prepared in the phagemid cloningvector λZAPII (Stratagene, LaJolla, Calif.) This library was screenedwith a DNA probe corresponding to nucleotides 471 to 1760 of theDrosGluCl cDNA (Cully et al., 1996, J Biol. Chem. 271: 20187-20191;accession number U58776) which codes for all but the last four aminoacids of the Drosophila glutamate-gated chloride channel. Two positiveclones, F5A and F6 were chosen for further analysis. These cDNA cloneswere shown to encode a truncated polypeptide disclosed in FIG. 4 and SEQID NO:4, referred to within this specification as trCfGluCl-1. It isshown in this specification that the truncation at the amino-terminalregion of clone F5A produced a frame shift mutation. It is also shown inthis specification that this truncation was in fact due to a deletion of71 nucleotides at the presumptive amino-terminal extracellular domain,resulting in a frame shift mutation that resulted in expression of thetruncated protein, trCfGluCl-1. A cDNA fragment containing the missingportion of a putative C. felis GluCl channel cDNA was generated by PCRamplification of randomly primed flea cDNA. Primer-1(5′-CTCAGAGTCAGGATCCGGCTA-3′; SEQ ID NO:5) and Primer-2(5′-CTGAAAGTTAACTGGACACTG-3′; SEQ ED NO:6) were used in a standard PCRreaction to amplify a 532 bp PCR fragment that was shown by DNA sequenceanalysis to contain the missing 71 nucleotides and flanking sequencesdisclosed in the F5A clone. A 517 bp BamHI/HpaI fragment of this PCRproduct was isolated and inserted into a BamHI/HpaI digested F5A cloneto generate the full length cDNA clone designated Flea51, as shown inFIGS. 1A-B. This cDNA molecule contains an open reading frame whichencodes a C. felis GluCl channel, as shown in FIG. 2, as set forth asSEQ ID NO:2. In addition, the 5′ untranslated region the exemplifiedcDNA which encodes a CfGluCl channel protein was determined and ispresented as SEQ ID NO:7.

A variety of mammalian expression vectors may be used to express arecombinant C. felis GluCl channel protein in mammalian cells.Expression vectors are defined herein as DNA sequences that are requiredfor the transcription of cloned DNA and the translation of their mRNAsin an appropriate host. Such vectors can be used to express eukaryoticDNA in a variety of hosts such as bacteria, blue green algae, plantcells, insect cells and animal cells. Specifically designed vectorsallow the shuttling of DNA between hosts such as bacteria-yeast orbacteria-animal cells. An appropriately constructed expression vectorshould contain: an origin of replication for autonomous replication inhost cells, selectable markers, a limited number of useful restrictionenzyme sites, a potential for high copy number, and active promoters. Apromoter is defined as a DNA sequence that directs RNA polymerase tobind to DNA and initiate RNA synthesis. A strong promoter is one whichcauses mRNAs to be initiated at high frequency. Expression vectors mayinclude, but are not limited to, cloning vectors, modified cloningvectors, specifically designed plasmids or viruses.

Commercially available mammalian expression vectors which may besuitable for recombinant C. felis GluCl channel protein expression,include but are not limited to, pcDNA3.1 (Invitrogen), pLITMUS28,pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI,pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXT1(Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2)(ATCC 37110), pdBPV-MM heo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199),pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), andλZD35 (ATCC 37565).

A variety of bacterial expression vectors may be used to express arecombinant C. felis GluCl channel protein in bacterial cells.Commercially available bacterial expression vectors which may besuitable for recombinant C. felis GluCl channel protein expressioninclude, but are not limited to pCR2.1 (Invitrogen), pET11a (Novagen),lambda gt11 (Invitrogen), and pKK223-3 (Pharmacia).

A variety of fungal cell expression vectors may be used to expressrecombinant C. felis GluCl channel protein in fungal cells. Commerciallyavailable fungal cell expression vectors which may be suitable forrecombinant C. felis GluCl channel expression include but are notlimited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).

A variety of insect cell expression vectors may be used to expressrecombinant receptor in insect cells. Commercially available insect cellexpression vectors which may be suitable for recombinant expression of aC. felis GluCl channel protein include but are not limited topBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).

An expression vector containing DNA encoding a C. felis GluCl channelprotein and/or C. felis GluCl channel-like protein may be used forexpression of C. felis GluCl channel protein in a recombinant host cell.Recombinant host cells may be prokaryotic or eukaryotic, including butnot limited to bacteria such as E. coli, fungal cells such as yeast,mammalian cells including but not limited to cell lines of human,bovine, porcine, monkey and rodent origin, and insect cells includingbut not limited to Drosophila- and silkworm-derived cell lines. Celllines derived from mammalian species which may be suitable and which arecommercially available, include but are not limited to, L cellsL-M(TK^(—)) (ATCC CCL 1.3), L cells LM (ATCC CCL 1.2), Saos-2 (ATCCHTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70),COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-KI (ATCC CCL 61), 3T3(ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCCCRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL209).

The cloned human C. felis GluCl channel cDNA obtained through themethods described above may be recombinantly expressed by molecularcloning into an expression vector (such as pcDNA3.1, pCR2.1,pBlueBacHis2 and pLITMUS28) containing a suitable promoter and otherappropriate transcription regulatory elements, and transferred intoprokaryotic or eukaryotic host cells to produce recombinant C. felisGluCl channel protein. Techniques for such manipulations can be founddescribed in Sambrook, et al., supra, are discussed at length in theExample section and are well known and easily available to the artisanof ordinary skill in the art.

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to direct injection,transformation, transfection, protoplast fusion, lipofection, andelectroporation. The expression vector-containing cells are clonallypropagated and individually analyzed to determine whether they produceC. felis GluCl protein. Identification of C. felis GluCl expressing hostcell clones may be done by several means, including but not limited toimmunological reactivity with anti-C. felis GluCl antibodies, and thepresence of host cell-associated GluCl activity.

Expression of C. felis GluCl DNA may also be performed using in vitroproduced synthetic mRNA. Synthetic mRNA can be efficiently translated invarious cell-free systems, including but not limited to wheat germextracts and reticulocyte extracts, as well as efficiently translated incell based systems, including but not limited to microinjection intofrog oocytes, with microinjection into frog oocytes being preferred.

To determine the C. felis GluCl channel cDNA sequence(s) that yieldsoptimal levels of C. felis GluCl channel protein, cDNA moleculesincluding but not limited to the following can be constructed: a cDNAfragment containing the full-length open reading frame for C. felisGluCl channel protein as well as various constructs containing portionsof the cDNA encoding only specific domains of the protein or rearrangeddomains of the protein. All constructs can be designed to contain none,all or portions of the 5′ and/or 3′ untranslated region of a C. felisGluCl channel cDNA. The expression levels and activity of C. felis GluClchannel protein can be determined following the introduction, bothsingly and in combination, of these constructs into appropriate hostcells. Following determination of the C. felis GluCl channel cDNAcassette yielding optimal expression in transient assays, this C. felisGluCl channel cDNA construct is transferred to a variety of expressionvectors (including recombinant viruses), including but not limited tothose for expression in host cells including, but not limited to,mammalian cells, insect cells such as baculovirus-infected insect cells,oocytes such as Xenopus oocytes, bacterial such asE. coli, and the yeastS. cerevisiae.

The present invention also relates to methods of expressing an active C.felis GluCl channel protein and biological equivalents disclosed herein,assays employing these recombinantly expressed gene products, cellsexpressing these gene products, and agonistic and/or antagonisticcompounds identified through the use of assays utilizing theserecombinant forms, including, but not limited to, one or more modulatorsof a C. felis GluCl channel.

A preferred expression system for the electrophysiological-based assaysand related improved methods of measuring glutamate-gated chloridechannel activity and modulation comprise injecting nucleic acidmolecules into Xenopus laevis oocytes. The general use of Xenopusoocytes in the study of ion channel activity is known in the art(Dascal, 1987, Crit. Rev. Biochem. 22: 317-317; Lester, 1988, Science241: 1057-1063; see also Methods of Enzymology, Vol. 207, 1992, Ch.14-25, Rudy and Iverson, ed., Academic Press, Inc., New York). A portionof the present invention discloses an improved method of measuringchannel activity and modulation by agonists and/or antagonists which isseveral-fold more sensitive than previously disclosed. The Xenopusoocytes are injected with nucleic acid material, including but notlimited to DNA, mRNA or cRNA which encode a gated-channel, whereinchannel activity may be measured as well as response of the channel tovarious modulators. To this end, the present invention relates to animproved in vitro method of measuring ion channel activity in eukaryoticcells, especially Xenopus oocytes, which comprises utilizing a holdingpotential more positive than the reversal potential for chloride (i.e,greater than −30 mV), preferably about 0 mV. This alteration in assaymeasurement conditions has resulting in a 10-fold increase insensitivity of the assay to modulation by ivermectin phosphate.Therefore, this improved assay will allow screening and selecting forcompounds which modulate GluCl activity at levels which were previouslythought to be undetectable. Data is presented in Example Section 2 whichexemplifies the use of this improved assay for detecting expressed ionchannel activity in Xenopus oocytes. It will be evident to the skilledartisan that this improved method may be utilized in various ion channelmeasurement assays, and especially assays which measure glutamate-gatedactivity in a eukaryotic cell, such as a Xenopus oocyte. It isespecially preferred that invertebrate glutamate-gated chloridechannels, including but in not way limited to Caenorhabditis elegans,Drosophila melonogaster and Ctenocephalides felis glutamate-gatedchannel proteins, be utilized in an assay to screen and select forcompounds which modulate the activity of these channels.

Levels of C. felis GluCl protein in host cells are quantitated byimmunoaffinity and/or ligand affinity techniques. Cells expressing GluClcan be assayed for the number of GluCl molecules expressed by measuringthe amount of radioactive glutamate or ivermectin binding to cellmembranes. GluCl-specific affinity beads or GluCl-specific antibodiesare used to isolate for example ³⁵S-methionine labelled or unlabelledGluCl protein. Labelled GluCl protein is analyzed by SDS PAGE.Unlabelled GluCl protein is detected by Western blotting, ELISA or RIAassays employing GluCl specific antibodies.

Recombinant C. felis GluCl channel protein can be separated from othercellular proteins by use of an immunoaffinity column made withmonoclonal or polyclonal antibodies specific for full-length C. felisGluCl channel protein, or polypeptide fragments of C. felis GluClchannel protein. Additionally, polyclonal or monoclonal antibodies maybe raised against a synthetic peptide (usually from about 9 to about 25amino acids in length) from a portion of the protein as disclosed in SEQID NO:2. Monospecific antibodies to C. felis GluCl channel are purifiedfrom mammalian antisera containing antibodies reactive against a C.felis GluCl channel or are prepared as monoclonal antibodies reactivewith aC. fetis GluCl channel using the technique of Kohler and Milstein(1975, Nature 256: 495-497). Monospecific antibody as used herein isdefined as a single antibody species or multiple antibody species withhomogenous binding characteristics for aC. felis GluCl channel.Homogenous binding as used herein refers to the ability of the antibodyspecies to bind to a specific antigen or epitope, such as thoseassociated with a C. felis GluCl channel, as described above. C. felisGluCl channel protein-specific antibodies are raised by immunizinganimals such as mice, rats, guinea pigs, rabbits, goats, horses and thelike, with an appropriate concentration of C. felis GluCl channelprotein or a synthetic peptide generated from a portion of C. felisGluCl channel with or without an immune adjuvant. Therefore, the presentinvention also relates to polyclonal and monoclonal antibodies raised inresponse to the C. felis GluCl channel protein disclosed herein, or abiologically active fragment thereof.

Preimmune serum is collected prior to the first immunization. Eachanimal receives between about 0.1 μg and about 1000 μg of C. felis GluClchannel protein associated with an acceptable immune adjuvant. Suchacceptable adjuvants include, but are not limited to, Freund's complete,Freund's incomplete, alum-precipitate, water in oil emulsion containingCorynebacterium parvum and tRNA. The initial immunization consists of C.felis GluCl channel protein or peptide fragment thereof in, preferably,Freund's complete adjuvant at multiple sites either subcutaneously (SC),intraperitoneally (IP) or both. Each animal is bled at regularintervals, preferably weekly, to determine antibody titer. The animalsmay or may not receive booster injections following the initialimmunization. Those animals receiving booster injections are generallygiven an equal amount of C. felis GluCl channel in Freund's incompleteadjuvant by the same route. Booster injections are given at about threeweek intervals until maximal titers are obtained. At about 7 days aftereach booster immunization or about weekly after a single immunization,the animals are bled, the serum collected, and aliquots are stored atabout −20° C.

Monoclonal antibodies (mAb) reactive with C. felis GluCl channel areprepared by immunzing inbred mice, preferably Balb/c, with C. felisGluCl channel protein. The mice are immunized by the IP or SC route withabout 1 μg to about 100's, preferably about 10's, of C. felis GluClchannel protein in about 0.5 ml buffer or saline incorporated in anequal volume of an acceptable adjuvant, as discussed above. Freund'scomplete adjuvant is preferred. The mice receive an initial immunizationon day 0 and are rested for about 3 to about 30 weeks. Immunized miceare given one or more booster immunizations of about 1 to about 100's ofC. felis GluCl channel protein in a buffer solution such as phosphatebuffered saline by the intravenous (I) route. Lymphocytes, from antibodypositive mice, preferably splenic lymphocytes, are obtained by removingspleens from immunized mice by standard procedures known in the art.Hybridoma cells are produced by mixing the splenic lymphocytes with anappropriate fusion partner, preferably myeloma cells, under conditionswhich will allow the formation of stable hybridomas. Fusion partners mayinclude, but are not limited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11;S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibody producingcells and myeloma cells are fused in polyethylene glycol, about 1000mol. wt., at concentrations from about 30% to about 50%. Fused hybridomacells are selected by growth in hypoxanthine, thymidine and aminopterinsupplemented Dulbecco's Modified Eagles Medium (DMEM) by proceduresknown in the art. Supernatant fluids are collected form growth positivewells on about days 14, 18, and 21 and are screened for antibodyproduction by an immunoassay such as solid phase immunoradioassay(SPIRA) using C. felis GluCl channel protein as the antigen. The culturefluids are also tested in the Ouchterlony precipitation assay todetermine the isotype of the mAb. Hybridoma cells from antibody positivewells are cloned by a technique such as the soft agar technique ofMacPherson, 1973, Soft Agar Techniques, in Tissue Culture Methods andApplications, Kruse and Paterson, Eds., Academic Press.

Monoclonal antibodies are produced in vivo by injection of pristineprimed Balb/c mice, approximately 0.5 ml per mouse, with about 2×10⁶ toabout 6×10⁶ hybridoma cells about 4 days after priming. Ascites fluid iscollected at approximately 8-12 days after cell transfer and themonoclonal antibodies are purified by techniques known in the art.

In vitro production of anti-C. felis GluCl channel protien mAb iscarried out by growing the hybridoma in DMEM containing about 2% fetalcalf serum to obtain sufficient quantities of the specific mAb. The mAbare purified by techniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of C. felisGluCl channel protein in body fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for C. felis GluCl channel peptidefragments, or full-length C. felis GluCl channel protein.

C. felis GluCl channel antibody affinity colulmns are made, for example,by adding the antibodies to Affigel-10 (Biorad), a gel support which ispre-activated with N-hydroxysucciiimde esters such that the antibodiesform covalent linkages with the agarose gel bead support. The antibodiesare then coupled to the gel via amide bonds with the spacer arm. Theremaining activated esters are then quenched with 1M ethanolamine HCl(pH 8). The column is washed with water followed by 0.23M glycine HCl(pH 2.6) to remove any non-conjugated antibody or extraneous protein.The column is then equilibrated in phosphate buffered saline (pH 7.3)and the cell culture supernatants or cell extracts containingfull-length C. felis GluCl channel protein or C. felis GluCl channelprotein fragments are slowly passed through the column. The column isthen washed with phosphate buffered saline until the optical density(A280) falls to background, then the protein is eluted with 0.23Mglycine-HCl (pH 2.6). The purified C. felis GluCl channel protein isthen dialyzed against phosphate buffered saline.

Levels of C. felis GluCl channel protein in host cells is quantified bya variety of techniques including, but not limited to, immunoaffinityand/or ligand affinity techniques. C. felis GluCl channelprotein-specific affinity beads or C. felis GluCl channelprotein-specific antibodies are used to isolate ³⁵S-methionine labeledor unlabelled C. felis GluCl channel protein. Labeled C. felis GluClchannel protein is analyzed by SDS-PAGE. Unlabelled C. felis GluClchannel protein is detected by Western blotting, ELISA or RIA assaysemploying C. felis GluCl channel protein specific antibodies.

Following expression of C. felis GluCl channel protein in a host cell,C. felis GluCl channel protein may be recovered to provide C. felisGluCl channel protein in active form. Several C. felis GluCl channelprotein purification procedures are available and suitable for use.Recombinant C. felis GluCl channel protein may be purified from celllysates and extracts, or from conditioned culture medium, by variouscombinations of, or individual application of salt fractionation, ionexchange chromatography, size exclusion chromatography, hydroxylapatiteadsorption chromatography and hydrophobic interaction chromatography. Itis also possible to prepare membrane preparations from a recombinanthost cell which contains a recombinant vector which expresses an activeC. felis GluCl channel. Such membrane preparations from recombinantcells will be useful for in vitro-based screening assays for compoundswhich modulate C. felis GluCl channel activity.

Compounds identified according to the methods disclosed herein may beused alone at appropriate dosages defined by routine testing in order toobtain optimal inhibition of the GluCl receptor or its activity whileminimizing any potential toxicity. In addition, co-administration orsequential administration of other agents may be desirable.

The method of the present invention also has the objective of providingsuitable topical, oral, systemic and parenteral pharmaceuticalformulations for use in the novel methods of treatment of the presentinvention. The compositions containing compounds identified according tothis invention as the active ingredient for use in the modulation ofGluCl receptors can be administered in a wide variety of therapeuticdosage forms in conventional vehicles for administration. For example,the compounds can be administered in such oral dosage forms as tablets,capsules (each including timed release and sustained releaseformulations), pills, powders, granules, elixirs, tinctures, solutions,suspensions, syrups and emulsions, or by injection. Likewise, they mayalso be administered in intravenous (both bolus and infusion),intraperitoneal, subcutaneous, topical with or without occlusion, orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts. An effective but non-toxic amount ofthe compound desired can be employed as a GluCl modulating agent.

The daily dosage of the products may be varied over a wide range from0.001 to 1,000 mg per patient, per day. For oral administration, thecompositions are preferably provided in the form of scored or unscoredtablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, and 50.0 milligrams of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. An effectiveamount of the drug is ordinarily supplied at a dosage level of fromabout 0.0001 mg/kg to about 100 mg/kg of body weight per day. Thedosages of the GluCl receptor modulators are adjusted when combined toachieve desired effects. On the other hand, dosages of these variousagents may be independently optimized and combined to achieve asynergistic result wherein the pathology is reduced more than it wouldbe if either agent were used alone.

Advantageously, compounds active in the method of the present inventionmay be administered in a single daily dose, or the total daily dosagemay be administered in divided doses of two, three or four times daily.Furthermore, compounds active in the method of the present invention canbe administered in intranasal form via topical use of suitableintranasal vehicles, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in thatart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds active in the method of thepresent invention is selected in accordance with a variety of factorsincluding type, species, age, weight, sex and medical condition of thepatient; the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

In the methods of the present invention, the compounds active thereincan form the active ingredient, and are typically administered inadmixture with suitable pharmaceutical diluents, excipients or carriers(collectively referred to herein as “carrier” materials) suitablyselected with respect to the intended form of administration, that is,oral tablets, capsules, elixirs, syrups and the like, and consistentwith conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methyl-cellulose and thelike. Other dispersing agents which may be employed include glycerin andthe like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations which generally containsuitable preservatives are employed when intravenous administration isdesired.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as,e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, PPG2 myristyl propionate, and the like, to form,e.g., alcoholic solutions, topical cleansers, cleansing creams, skingels, skin lotions, and shampoos in cream or gel formulations.

The compounds active in the method of the present invention can also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine or phosphatidylcholines.

Compounds active in the method of the present invention may also bedelivered by the use of monoclonal antibodies as individual carriers towhich the compound molecules are coupled. The compounds active in themethod of the present invention may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinyl-pyrrolidone, pyran copolymer,polyhydroxy-propylmethacryl-amidephenol,polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds activein the method of the present invention may be coupled to a class ofbiodegradable polymers useful in achieving controlled release of a drug,for example, polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydro-pyrans,polycyanoacrylates and cross-linked or amphipathic block copolymers ofhydrogels.

The compounds that are active in the methods of the present inventionare useful as antiparastic agents against endo and ecto parasites,particularly helminths and arthropods, which cause numerous parasiticdiseases in humans, animals, and plants.

Parasitic diseases may be caused by either endoparasites orectoparasites. Endoparasites are those parasites which live inside thebody of the host, either within an organ (such as the stomach, lungs,heart, intestines, etc.) or simply under the skin. Ectoparasites arethose parasites which live on the outer surface of the host but stilldraw nutrients from the host.

The endoparasitic diseases generally referred to as helminthiasis aredue to infection of the host with parasitic worms known as helminths.Helminthiasis is a prevalent and serious worldwide economic problem dueto infection of domesticated animals such as swine, sheep, horses,cattle, goats, dogs, cats, and poultry. Many of these infections arecaused by the group of worms described as nematodes which cause diseasesin various species of animals throughout the world. These diseases arefrequently serious and can result in the death of the infected animal.The most common genera of nematodes infecting the animas referred toabove are Haemonchus, Trichostrongylus, Ostertagia, Nematodirus,Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia, Trichuris,Strongylus, Trichonema, Dictyocaulus, Capillaria, Heterakis, Toxocara,Ascaridia, Oxyuris, Ancylostoma, Uncinaria, Toxascaris, and Parascaris.Many parasites are species specific (infect only one host) and most alsohave a preferred site of infection within the animal. Thus Haemonchusand Ostertagia primarily infect the stomach while Nematodirus andCooperia mostly attack the intestines. Other parasites prefer to residein the heart, eyes, lungs, blood vessels, and the like while stillothers are subcutaneous parasites. Helminthiasis can lead to weakness,weight loss, anemia, intestinal damage, malnutrition, and damage toother organs. If left untreated these diseases can result in the deathof the animal.

Diseases caused by ectoparasitic arthropods such as ticks, mites, lice,stable flies, hornflies, blowflies, fleas, and other biting insects suchas Tenophalides, Ixodes, Psoroptes, Lucilia, and Hemotobia, are also aserious problem. hafection and infestation by these parasites results inloss of blood, skin lesions, and can interfere with normal eating habitsthus causing weight loss. These infections can also result intransmission of serious diseases such as encephalitis, anaplasmosis,swine pox, and the like which can be fatal. The compounds that areactive in the method disclosed herein are useful for the prevention andtreatment of these infections and infestations.

Animals may be infected by several species of parasite at the same timesince infection by one parasite may weaken the animal and make it moresusceptible to infection by a second species of parasite. Thus acompound with a broad spectrum of activity is particularly advantageousin the treatment of these diseases. The compounds of this invention haveactivity against these parasites, and in addition are also activeagainst Dirofilaria in dogs, Nematospiroides and Syphacia in rodents,biting insects, and migrating diperous larvae such as Hypoderma sp. incattle, and Gastrophilus in horses.

The compounds active in the method disclosed herein are also usefulagainst endo and ecto parasites which cause parasitic diseases inhumans. Examples of such endoparasites which infect man includegastrointestinal parasites of the genera Ancylostoma, Necator, Ascaris,Strongyloides, Trichinella, Capillaria, Trichuris, Enterobius, and thelike. Other endoparasites which infect man are found in the blood or inother organs. Examples of such parasites are the filarial wormsWucheria, Brugia, Onchocerca, and the like as well as extra-intestinalstages of the intestinal worms Strongylides and Trichinella.Ectoparasites which parasitize man include arthropods such as ticks,fleas, mites, lice, and the like and, as with domestic animals,infections by these parasites can result in transmission of serious andeven fatal diseases. The active compounds are active against these endoand ecto parasites and in addition are also active against bitinginsects and other dipterous pests which annoy humans.

The compounds active in the method disclosed herein are also usefulagainst common household pests such as Blatella sp. (cockroach), Tineolasp. (clothes moth), Attagenus sp. (carpet beetle), Musca domestica(housefly) and against Solenopsis Invicta (imported fire ant).

The compounds active in the method disclosed herein are furthermoreuseful against agricultural pests such as aphids (Acyrthiosiphon sp.),locusts, spider mites, and boll weevils as well as against insect pestswhich attack stored grains such as Tribolium sp. and Tenebrio sp., andagainst immature stages of insects living on plant tissue. The compoundsare also useful as a nematodicide for the control of soil nematodes andplant parasites such as Meloidogyne sp., which may be agriculturallyimportant.

For use as an antiparasitic agent in animals the compounds may beadministered internally either orally, or by injection, or topically asa liquid drench or as a shampoo.

For oral administration, the compounds active in the method disclosedherein may be administered in capsule, tablet, or bolus form oralternatively they can be mixed in the animals feed. The capsules,tablets, and boluses are comprised of the active ingredient incombination with an appropriate carrier vehicle such as starch, talc,magnesium stearate, or di-calcium phosphate. These unit dosage forms areprepared by intimately mixing the active ingredient with suitablefinely-powdered inert ingredients including diluents, fillers,disintegrating agents, and/or binders such that a uniform mixture isobtained. An inert ingredient is one that will not react with thecompounds and which is non-toxic to the animal being treated. Suitableinert ingredients include starch, lactose, talc, magnesium stearate,vegetable gums and oils, and the like. These formulations may contain awidely variable amount of the active and inactive ingredients dependingon numerous factors such as the size and type of the animal species tobe treated and the type and severity of the infection. The activeingredient may also be administered as an additive to the feed by simplymixing the compound with the feedstuff or by applying the compound tothe surface of the feed. Alternatively the active ingredient may bemixed with an inert carrier and the resulting composition may theneither be mixed with the feed or fed directly to the animal. Suitableinert carriers include corn meal, citrus meal, fermentation residues,soya grits, dried grains and the like. The active ingredients areintimately mixed with these inert carriers by grinding, stirring,milling, or tumbling such that the final composition contains from 0.001to 5% by weight of the active ingredient.

The compounds active in the method disclosed herein may alternatively beadministered parenterally via injection of a formulation consisting ofthe active ingredient dissolved in an inert liquid carrier. Injectionmay be either intramuscular, intramural, intratracheal, or subcutaneous.The injectable formulation consists of the active ingredient mixed withan appropriate inert liquid carrier. Acceptable liquid carriers includethe vegetable oils such as peanut oil, cotton seed oil, sesame oil andthe like as well as organic solvents such as solketal, glycerol formaland the like. As an alternative, aqueous parenteral formulations mayalso be used. The vegetable oils are the preferred liquid carriers. Theformulations are prepared by dissolving or suspending the activeingredient in the liquid carrier such that the final formulationcontains from 0.005 to 10% by weight of the active ingredient.

Topical application of the compounds active in the method disclosedherein is possible through the use of a liquid drench or a shampoocontaining the instant compounds as an aqueous solution, dispersion orsuspension. These formulations generally contain a suspending agent suchas bentonite, a wetting agent or the like excipient, and normally willalso contain an antifoaming agent. Formulations containing from 0.001 to1% by weight of the active ingredient are acceptable. Preferredformulations are those containing from 0.01 to 1% by weight of theactive compounds.

The compounds active in the method disclosed herein are primarily usefulas antiparasitic agents for the treatment and/or prevention ofhelminthiasis in domestic animals such as cattle, sheep, horses, dogs,cats, goats, swine, and poultry. They are also useful in the preventionand treatment of parasitic infections of these animals by ectoparasitessuch as ticks, mites, lice, fleas and the like. They are also effectivein the treatment of parasitic infections of humans. In treating suchinfections the compounds may be used individually or in combination witheach other or with other unrelated antiparasitic agents. The dosage ofthe compounds required for best results depends on several factors suchas the species and size of the animal, the type and severity of theinfection, the method of administration and the compound used. Oraladministration of the compounds at a dose level of from 0.0005 to 10 mgper kg of animal body weight, either in a single dose or in severaldoses spaced a few days apart, generally gives good results. A singledose of one of the compounds normally gives excellent control howeverrepeat doses may be given to combat re-infection or for parasite specieswhich are unusually persistent. The techniques for administering thesecompounds to animals are known to those skilled in the veterinary field.

The compounds active in the method disclosed herein may also be used tocombat agricultural pests which attack crops either in the field or instorage. The compounds are applied for such uses as sprays, dusts,emulsions and the like either to the growing plants or the harvestedcrops. The techniques for applying these compounds in this manner areknown to those skilled in the agricultural arts.

Pharmaceutically useful compositions comprising modulators of the C.felis GluCl channel may be formulated according to known methods such asby the admixture of a pharmaceutically acceptable carrier. Examples ofsuch carriers and methods of formulation may be found in Remington'sPharmaceutical Sciences.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

The present invention is also directed to methods for screening forcompounds which modulate the expression of DNA or RNA encoding C. felisGluCl as well as the function of the C. felis GluCl protein in vivo.Compounds which modulate these activities may be DNA, RNA, peptides,proteins, or non-proteinaceous organic molecules. Compounds may modulateby increasing or attenuating the expression of DNA or RNA encoding C.felis GluCl, or the function of the C. felis GluCl protein. Compoundsthat modulate the expression of DNA or RNA encoding C. felis GluCl orthe function of C. felis GluCl protein may be detected by a variety ofassays. The assay may be a simple “yes/no” assay to determine whetherthere is a change in expression or function. The assay may be madequantitative by comparing the expression or function of a test samplewith the levels of expression or function in a standard sample.Modulators identified in this process are useful as therapeutic agents,insecticides and anthelminthics.

Kits containing C. felis GluCl DNA, antibodies to C. felis GluCl, or C.felis GluCl protein may be prepared. Such kits are used to detect DNAwhich hybridizes to C. felis GluCl DNA or to detect the presence of C.felis GluCl protein or peptide fragments in a sample. Suchcharacterization is useful for a variety of purposes including but notlimited to forensic analyses and epidemiological studies.

The DNA molecules, RNA molecules, recombinant protein and antibodies ofthe present invention may be used to screen and measure levels of C.felis GluCl DNA, RNA or protein. The recombinant proteins, DNAmolecules, RNA molecules and antibodies lend themselves to theformulation of kits suitable for the detection and typing of C. felisGluCl. Such a kit would comprise a compartmentalized carrier suitable tohold in close confinement at least one container. The carrier wouldfurther comprise reagents such as recombinant C. felis GluCl protein oranti-GluCl antibodies suitable for detecting GluCl. The carrier may alsocontain a means for detection such as labeled antigen or enzymesubstrates or the like.

Nucleotide sequences that are complementary to the C. felis GluClencoding DNA sequence can be synthesized for antisense therapy. Theseantisense molecules may be DNA, stable derivatives of DNA such asphosphorothioates or methylphosphonates, RNA, stable derivatives of RNAsuch as 2′-O-alkylRNA, or other GluCl antisense oligonucleotidemimetics. C. felis GluCl antisense molecules may be introduced intocells by microinjection, liposome encapsulation or by expression fromvectors harboring the antisense sequence. C. felis GluCl antisensetherapy may be particularly useful for the treatment of diseases whereit is beneficial to reduce GluCl activity.

C. felis GluCl DNA may be used to introduce GluCl into the cells oftarget organisms. The GluCl gene can be ligated into viral vectors whichmediate transfer of the GluCl DNA by infection of recipient host cells.Suitable viral vectors include retrovirus, adenovirus, adeno-associatedvirus, herpes virus, vaccinia virus, polio virus and the like.Alternatively, GluCl DNA can be transferred into cells by non-viraltechniques including receptor-mediated targeted DNA transfer usingligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofectionmembrane fusion or direct microinjection. These procedures andvariations thereof are suitable for ex vivo as well as in vivo GluClgene therapy. GluCl gene therapy may be particularly useful where it isbeneficial to elevate GluCl activity.

The present invention also provides for improved methods of screeningfor modulators of a GluCl channel in general and modulators of the C.felis GluCl channel in particular. It is shown in Example Section 2 thatimproved assay conditions result in a 10-fold increase in channelmodulator sensitivity when compared to previous known assay conditions.In a preferred aspect of measuring GluCl channel activity, oocytes areinjected with synthetic RNAs or DNAs for one or more C. felis GluClproteins. Following an appropriate period of time to allow forexpression, GluCl activity is measured by specific ligand binding andelectrophysiological characteristics of the host cells expressing GluClDNA. Voltage-clamp studies were conducted as described in ExampleSection 2, preferably utilizing a holding potential of 0 mV duringmeasurements of modulation by ivermectin phosphate. Exemplification ofthis improved method of a cell-based assay of GluCl channel activity isshown in Example Section 2 and is further detailed in FIGS. 5A and 5B(showing activation of CfGluCl-1 by glutamate), FIG. 6 (showing that theCfGluCl-1 channel is selective for chloride), and FIGS. 7A and 7B(showing that IVM-PO₄ is an agonist of a GluCl channel).

The following examples are provided to illustrate the present inventionwithout, however, limiting the same hereto.

EXAMPLE 1 Isolation and Characterization of a Full Length cDNA Encodinga Ctenocephalides felis GluCl Channel

Ctenocephalides felis Poly A⁺ RNA isolation—Poly(A)⁺ RNA was preparedfrom whole fleas. The fleas were rapidly frozen in liquid N₂ and groundwith a mortar and pestle while submerged in liquid N₂. The frozen,powdered C. felis tissue was added to a solution containing 4Mguanidinium thiocyanate, 5 mM sodium citrate pH 7.0, and 0.1Mβ-mercaptoethanol (1 gm tissue/10 ml solution), and was mixed with apolytron homogenizer. After 1 minute of homogenization, 0.5% sodiumsarkosyl was added and mixed well and the solution was centrifuged at10,000 rpm for 10 minutes. The supernatant was layered over a 5.7M CsClcushion and centrifuged for 18 hours at 33,000 rpm. The RNA pellet waswashed with 70% ethanol, resuspended in H₂O and extracted withchloroform:isobutanol, 4:1 and precipitated with ethanol. Poly (A)⁺ RNAwas isolated by two rounds of purification on oligo (dT)-cellulosecolumns.

Isolation of a cDNA Partially Encoding a C.felis GluCl Channel—Anoligo-dT primed C. felis cDNA library was prepared in the phagemidcloning vector λZAPII (Stratagene, LaJolla, Calif.) This library wastransfected into E. coli PLK-F′ cells, plated on NZY medium (Sambrook,et al, 1989, Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), andincubated 18 hrs. at 37° C. The resultant plaques were transferred toDurulose membranes (Stratagene). The membranes were prehybridized in 50%formamide; 2× Denhardts solution; 5× SSPE; 0.1%SDS; 100 μg/ml solmonsperm DNA for 16 hours and hybridized in the above prehybridizationsolution containing 10% dextran sulfate for 24 hours with 2×10⁷ cpm ahybridization probe was a PCR-generated fragment of the DrosGluCl cDNAcorresponding to nucleotides 471-1760 of the cDNA as listed in GenBank(Cully, et al., 1996, J. Biol. Chem. 271(33): 20187-20191; accessionnumber U58776). This DNA codes for all but the last four amino acids ofthe mature Drosophila glutamate-gated chloride channel. The filters werewashed at 52° C. in 6×SSC, 0.1% SDS. The washed filters were exposed toX-ray film. Two positive clones, F5A and F6 were chosen for furtheranalysis. These clones were converted into plasmids by in vivo excisionas per the Stratagene protocol. Clone F5A was subjected to DNA sequenceanalysis and is disclosed in FIG. 3 and SEQ ID NO:3, and as follows:

ATGGACAGCA TTAGTTTGCT CCTACTTTTG ATAACATGTC TAAGTCTACA CACATGCTTATCTGCAAATG CAAAACCTCG TCTAGGAGGC GGCAAAGAAA ATTTCAGGGC CAAAGAAAAGCAAGTTCTGG ACCAAATTTT AGGCCCAGGC CATTACGATG CCAGAATAAG GCCTTCTGGAGTCAATGGAA CTGGAATACA GTGTCCAGTT AACTTTCAGG GAACAATGGC AGGATGAGAGGTTGAAATTT AACGACTTTG GAGGTCGTTT AAAATACTTA ACACTAACCG AAGCAAGTCGTGTATGGATG CCCGATTTGT TCTTTGCGAA TGAAAAGGAG GGCCACTTTC ACAACATCATCATGCCGAAC GTCTACATTC GTATTTTTCC TTACGGTTCC GTACTATACA GCATCAGGATATCGCTTACT TTGGCGTGTC CTATGAATCT GAAACTGTAT CCGCTCGATA GGCAGGTGTGCTCTCTCCGG ATGGCCAGTT ATGGTTGGAC CACAAACGAT CTGGTGTTTT TGTGGAAGGAAGGTGACCCG GTGCAGGTTG TCAAGAATCT ACATCTGCCC AGGTTTACGT TGGAGAAGTTCTTGACGGAT TATTGTAACA GCAAAACCAA TACCGGTGAA TACAGTTGCC TGAAGGTCGACCTGCTCTTT AAACGAGAGT TCTCGTACTA CCTGATCCAG ATCTACATTC CTTGTTGCATGTTGGTGATC GTTTCCTGGG TGTCGTTCTG GTTGGACCAG GGAGCGGTTC CGGCCAGAGTATCACTGGGT GTGACCACTC TCCTCACCAT GGCCACCCAG ACGTCGGGCA TAAACGCCTCCCTGCCGCCA GTGTCCTACA CAAAAGCCAT CGACGTCTGG ACCGGAGTCT GCCTCACGTTCGTCTTCGGG GCTTTGCTCG AATTCGCCCT CGTCAACTAC GCCTCCAGAT CCGATATGCACAGGGAAAAC ATGAAGAAAA AGCGCAGGGA ACTTGAACAA GCAGCCAGCC TGGACGCCGCCTCCGACCTG ATGGACGGCA CTGATGGCAC TTTTGCTATG AAGCCTCTGG TACGCCACTCCGTCGACGCC GTCGGTCTCG ATAAGGTTCG TCAGTGCGAG ATACACATGC AGCCGGCGTCCAGGCAGAAC TGCTGCAGGA GCTGGATAAG CAAATTCCCG ACGAGGTCGA AACGCATCGACGTCATATCA AGAATCACTT TCCCGCTGGT GTTTGCTTTG TTCAATCTGG TGTACTGGTCGACCTATTTG TTCAGGGACG AGGCGGAGGA GAATTAG (SEQ ID NO:3).

Clone F5A was shown to encode a truncated polypeptide disclosed in FIG.4 and SEQ ID NO:4, referred to within this specification as trCfGluCl-1,and disclosed as follows:

1  MDSISLLLLL ITCLSLHTCL SANAKPRLGG GKENFRAKEK QVLDQILGPG 51 HYDARIRPSGVNGTGIQCPV NFQGTMAG (SEQ ID NO:4).

Isolation of a cDNA Encoding a C. felis GluCl Channel—It was determinedthat clone F5A lacked an internal portion a possible C. felis GluClchannel cDNA at the presumptive amino-terminal extracellular domain,resulting in a frame shift mutation and the concomitant truncatedprotein, trCfGluCl-1. A cDNA fragment containing the missing portion ofa putative C. felis GluCl channel cDNA was generated by PCRamplification of randomly primed flea cDNA. Primer-1(CTCAGAGTCAGGATCCGGCTA; SEQ ID NO:5) and Primer-2(CTGAAAGTTAACTGGACACTG; SEQ ID NO:6) were used in a standard PCRreaction to amplify a 532 bp PCR fragment that was shown by DNA sequenceanalysis to contain the missing 71 nucleotides and flanking sequencesdisclosed in the F5A clone. This PCR fragment is as follows:

TCAGAGTCA G GATCC GGCTA TATTGGACGA TATGCTGCAT GGTCCCTGTC ATACAAATACTCCTTCGCCT TCACTGGAAC CAACCAAGAC TGTCCCCACG TGTCCGACAT CAGTTGAAGGAAATTCTGTG ACGACATGGC AACACTTTTG TTCAGGAACA ACAATAACAT CATCGACACAGAATATCGGC GAAGCCTATT CTTCGATTCA AGAAGAAGAA TTTCTTCACT TTATCTTCAGGGATGGACAG CATTAGTTTG CTCCTACTTT TGATAACATG TCTAAGTCTA CACACATGCTTATCTGCAAA TGCAAAACCT CGTCTAGGAG GCGGCAAAGA AAATTTCAGG GCCAAAGAAAAGCAAGTTCT GGACCAAATT TTAGGCCCAG GCCATTACGA TGCCAGAATA AGGCCTTCTGGAGTCAATGG AACTGGAGAC GGTCCGACCG TGGTAGCAGT CAACATCTAT CTGAGATCAATCAGCGAAAT AGATGACTAC AAAATGGAAT ACAGTGTCCA GTTAAC TTTC AG (SEQ ID NO:8)

This PCR fragment was cloned using the TA cloning vector kit(Invitrogen) and individual clones were sequenced to identify thoselacking PCR artifacts and containing the missing 71 bp fragment. A 517bp BamHI/HpaI fragment (Bam HI-GGATCC; HpaI GTTAAC, as underlined above)of this PCR product was isolated and inserted into a BamHI/HpaI digestedF5A clone (FIG. 3; SEQ ID NO:3) to generate the full length cDNA clonein the F5A pBS vector, designated Flea51, shown in FIG. 1, and set forthas SEQ ID NO:1 as follows:

ATGGACAGCA TTAGTTTGCT CCTACTTTTG ATAACATGTC TAAGTCTACA CACATGCTTATCTGCAAATG CAAAACCTCG TCTAGGAGGC GGCAAAGAAA ATTTCAGGGC CAAAGAAAAGCAAGTTCTGG ACCAAATTTT AGGCCCAGGC CATTACGATG CCAGAATAAG GCCTTCTGGAGTCAATGGAA CTGGAGACGG TCCGACCGTG GTAGCAGTCA ACATCTATCT GAGATCAATCAGCGAAATAG ATGACTACAA AATGGAATAC AGTGTCCAGT TAACTTTCAG GGAACAATGGCAGGATGAGA GGTTGAAATT TAACGACTTT GGAGGTCGTT TAAAATACTT AACACTAACCGAAGCAAGTC GTGTATGGAT GCCCGATTTG TTCTTTGCGA ATGAAAAGGA GGGCCACTTTCACAACATCA TCATGCCGAA CGTCTACATT CGTATTTTTC CTTACGGTTC CGTACTATACAGCATCAGGA TATCGCTTAC TTTGGCGTGT CCTATGAATC TGAAACTGTA TCCGCTCGATAGGCAGGTGT GCTCTCTCCG GATGGCCAGT TATGGTTGGA CCACAAACGA TCTGGTGTTTTTGTGGAAGG AAGGTGACCC GGTGCAGGTT GTCAAGAATC TACATCTGCC CAGGTTTACGTTGGAGAAGT TCTTGACGGA TTATTGTAAC AGCAAAACCA ATACCGGTGA ATACAGTTGCCTGAAGGTCG ACCTGCTCTT TAAACGAGAG TTCTCGTACT ACCTGATCCA GATCTACATTCCTTGTTGCA TGTTGGTGAT CGTTTCCTGG GTGTCGTTCT GGTTGGACCA GGGAGCGGTTCCGGCCAGAG TATCACTGGG TGTGACCACT CTCCTCACCA TGGCCACCCA GACGTCGGGCATAAACGCCT CCCTGCCGCC AGTGTCCTAC ACAAAAGCCA TCGACGTCTG GACCGGAGTCTGCCTCACGT TCGTCTTCGG GGCTTTGCTC GAATTCGCCC TCGTCAACTA CGCCTCCAGATCCGATATGC ACAGGGAAAA CATGAAGAAA AAGCGCAGGG AACTTGAACA AGCAGCCAGCCTGGACGCCG CCTCCGACCT GATGGACGGC ACTGATGGCA CTTTTGCTAT GAAGCCTCTGGTACGCCACT CCGTCGACGC CGTCGGTCTC GATAAGGTTC GTCAGTGCGA GATACACATGCAGCCGGCGT CCAGGCAGAA CTGCTGCAGG AGCTGGATAA GCAAATTCCC GACGAGGTCGAAACGCATCG ACGTCATATC AAGAATCACT TTCCCGCTGG TGTTTGCTTT GTTCAATCTGGTGTACTGGT CGACCTATTT GTTCAGGGAC GAGGCGGAGG AGAATTAG (SEQ ID NO:1).

This cDNA molecule contains an open reading frame which encodes a C.felis GluCl channel, as shown in FIG. 2, as set forth as SEQ ID NO:2,and as follows:

MDSISLLLLL ITCLSLHTCL SANAKPRLGG GKENFRAKEK QVLDQILGPG HYDARIRPSGVNGTGDGPTV VAVNIYLRSI SEIDDYKMEY SVQLTFREQW QDERLKFNDF GGRLKYLTLTEASRVWMPDL FFANEKEGHF HNIIMPNVYI RIFPYGSVLY SIRISLTLAC PMNLKLYPLDRQVCSLRMAS YGWTTNDLVF LWKEGDPVQV VKNLHLPRFT LEKFLTDYCN SKTNTGEYSCLKVDLLFKRE FSYYLIQIYI PCCMLVIVSW VSFWLDQGAV PARVSLGVTT LLTMATQTSGINASLPPVSY TKAIDVWTGV CLTFVFGALL EFALVNYASR SDMHRENMKK KRRELEQAASLDAASDLMDG TDGTFAMKPL VRHSVDAVGL DKVRQCEIHM QPASRQNCCR SWISKFPTRSKRIDVISRIT FPLVFALFNL VYWSTYLFRD EAEEN (SEQ ID NO:2).

In addition, the 5′ untranslated region the exemplified cDNA whichencodes a CfGluCl channel protein was determined and is presented as SEQID NO:7, and as follows:

AACTAGTGGA TCCCCCGGGC TGCAGGATTC GGCACGAGAA TTTTTTAAAA TAATCCTCAACAGCATGATA CAAGAGGATG ATTTTATGAT CCCTGTAAAC ACTTGCTTGA ATTTTAGATTGCAACTGGAG GCTCCGCTGA CACTCTCTCT TGTTCGAGCA CAGGAATTGC TCGACATCTGGTCAAACGCG GGCTACTTCA TAATATCCGA CGATGACAAT TTAATGTTCG GAGCAAGAACAATTGCAGAA TTTGAAGTGT ACTTTAACGA TACATTCGAA GGACGCATGA AAATGTGCACGATGTGCATG TTGCCCACCT TCTATTGACC AGCAAGCACC CCTTCGCCGG TGAGCATGTCACCCACCGAC AGGCGCCTTC TGTGCGCCCT CGACGACCTG CACTTAGCGG TTGCTAAGAAGCCCTAAGAA GCCGAGACGG TTCGCTTCGC CCGGGGGCGA TTCCTCACGA TGCACAAGCGGAGGCGCAAG AGGCTGACGA CGAGGAGCCT CAGAGTCAGG ATCCGGCTAT ATTGGACGATATGCTGCATG GTCCCTGTCA TACAAATACT CCTTCGCCTT CACTGGAACC AACCAAGACTGTCCCCACGT GTCCGACATC AGTTGAAGGA AATTCTGTGA CGACATGGCA ACACTTTTGTTCAGGAACAA CAATAACATC ATCGACACAG AATATCGGCG AAGCCTATTC TTCGATTCAAGAAGAAGAAT TTCTTCACTT TATCTTCAGG G (SEQ ID NO:7)

EXAMPLE 2 Expression of the CfGluCl-1 protein in Xenopus oocytes

The full-length cDNA encoding CfGluCl-1 in plasmid vector pBluescript(Stratagene, LaJolla, Calif.) is linearized and capped cRNA transcriptsare synthesized using appropriate oligonucleotide primers and themMESSAGE mMACHINE in vitro RNA transcription kit (Ambion). Xenopuslaevis oocytes were prepared and injected using standard methods asdescribed (Arena et al., 1991, Mol. Pharmacol. 40: 368-374; Arena et al,1992, Mol. Brain Res. 15: 339-348). Adult female Xenopus laevis wereanesthetized with 0.17% tricaine methanesulfonate and the ovaries weresurgically removed and placed in a dish consisting of (mM): NaCl 82.5,KCl 2, MgCl2 1, CaCl₂ 1.8, HEPES 5 adjusted to pH 7.5 with NaOH (OR-2).Ovarian lobes were broken open, rinsed several times, and gently shakenin OR-2 containing 0.2% collagenase (Sigma, Type IA) for 2-5 hours. Whenapproximately 50% of the follicular layers were removed, Stage V and VIoocytes were selected and placed in media consisting of (mM): NaCl 86,KCl 2, MgCl2 1, CaCl2 1.8, HEPES 5, Na pyruvate 2.5, theophylline 0.5,gentamicin 0.1 adjusted to pH 7.5 with NaOH (ND-96) for 2448 hoursbefore injection. For most experiments, oocytes were injected with 10 ngof cRNA in 50 nl of RNase free water. Control oocytes were injected with50 nl of water. Oocytes were incubated for 1-5 days in ND-96supplemented with 50 mg/ml gentamycin, 2.5 mM Na pyruvate and 0.5 mMtheophylline before recording. Incubations and collagenase digestionwere carried out at 180° C.

Voltage-clamp studies were conducted with the two microelectrode voltageclamp technique using a Dagan CA1 amplifier (Dagan Instruments,Minneapolis, Minn.). The current passing microelectrodes were filledwith 0.7M KCl plus 1.7M K₃-citrate and the voltage recordingmicroelectrodes were filled with 1.0M KCl. The extracellular solutionfor most experiments was saline consisting of (mM): NaCl 96, BaCl₂ 3.5,MgCl₂ 0.5, CaCl₂ 0.1, HEPES 5, adjusted to pH 7.5 with NaOH. Theextracellular chloride concentration was reduced in some experiments byequimolar replacement of NaCl with the sodium salt of the indicatedanion. Experiments were conducted at 21-24° C. Data were acquired usingthe program Pulse and most analysis was performed with the companionprogram Pulsefit (Instrutech Instruments, Great Neck, N.Y.) or with IgorPro (Wavemetrics, Lake Oswego, Oreg.). Data were filtered (fc, −3db) at1 kHz, unless otherwise indicated.

FIG. 5A and FIG. 5B show the activation of CfGluCl-1 by glutamate. FIG.5A shows superimposed current recordings in response to 10, 30, 100 and300 μM glutamate. The duration of exposure to glutamate is indicated bythe solid bar at top. FIG. 5B shoes the concentration-response curve forglutamate. Peak outward current is plotted vs. glutamate concentration.The solid curve is the best fit to the equationI/I_(max)={1+(EC₅₀/[glutamate])^(n)}⁻¹. For the experiment shown in FIG.5B, EC₅₀=9.3 μM, n=2.13. Agonists for other types of ligand-gatedchloride channels were also tested for the ability to activate

CfGluCl-1. GABA, glycine, histamine, acetylcholine and muscimol were allinactive.

FIG. 6 shows that the CfGluCl-1 channel is selective for chloride. Eachcurve represents the difference between the current measured with andwithout 10 μM glutamate. The voltage was ramped from −120 to +60 mV at 1volt/second. Chloride concentration was reduced from 104 mM to 8.2 mM byequimolar substitution of NaCl by Na-methanesulfonate or Na-gluconate.Each current-voltage relationship was fit to a seventh order polynomialusing non-linear least squares analysis and the reversal potential wastaken as the x-intercept of this polynomial. The reversal potentialmeasurements indicate that the relative permeability formethanesulfonate ( i.e., (permeability for methanesulfonate)l(permeability for chloride)) is 0.218 and the relative permeability forgluconate is 0.064.

FIGS. 7A and B show that ivermectin phosphate is an agonist of the fleaGluCl channel encoded by CfGluCl-1. FIG. 7A shows activation ofCfGluCl-1 by ivermectin phosphate (IVM-PO₄) and superimposed currentrecordings showing activation by 100 μM glutamate and 10 nM IVM-PO₄. Theactivation by IVM-PO₄ has a sigmoidal onset suggesting that multiplebinding sites must be occupied for opening. FIG. 7B shows theconcentration-response curve for IVM-PO₄. A single [IVM-PO₄] was testedon each oocyte. The ordinate is the maximal current induced by IVM-PO₄normalized by the peak current induced by 100 μM glutamate, a maximallyeffective concentration. The error bars indicate ±S.E.M. The holdingpotential was 0 mV for both sets of measurements. The filled circlesrepresent data for CfGluCl-1. The solid curve is the best fit to thisdata by

(1) I=I_(ivm,max)/{1+(EC₅₀/[IVM-PO₄])^(n)}

where I_(ivm,max)=0.718, EC₅₀=2.93 nM, and n=1.0. Also shown is thedose-response curve previously reported for the DmGluCl1 clone fromDrosophila metanogaster, except that in these earlier studies theholding potential was −80 mV (Cully et al., J. 1996, J. Biol. Chem. 271:20187-20191). This curve is the best fit to equation (1) formodification by IVM-PO₄, where I_(ivm,max)=0.35, EC₅₀=41 nM, and n=1.2.This data shows a 10-fold increase in potency. Additional data showsthat this increase in potency is not the result of differences betweenthe clones and/or in measurement technique. The measurements wererepeated on DmGluCl1 at a holding potential of 0 mV (filled squares);the solid curve is the best fit to equation (1) with the constraint thatthe EC₅₀ and n are the same as for CfGluCl1. The goodness of fitindicates that the EC₅₀ for DmGluCl1 is similar to that for CfGluCl1 andthat both channels are activated by WM-PO₄ at concentrations 10-foldlower than previously recognized.

8 1 1368 DNA ctenocephalides felis 1 atggacagca ttagtttgct cctacttttgataacatgtc taagtctaca cacatgctta 60 tctgcaaatg caaaacctcg tctaggaggcggcaaagaaa atttcagggc caaagaaaag 120 caagttctgg accaaatttt aggcccaggccattacgatg ccagaataag gccttctgga 180 gtcaatggaa ctggagacgg tccgaccgtggtagcagtca acatctatct gagatcaatc 240 agcgaaatag atgactacaa aatggaatacagtgtccagt taactttcag ggaacaatgg 300 caggatgaga ggttgaaatt taacgactttggaggtcgtt taaaatactt aacactaacc 360 gaagcaagtc gtgtatggat gcccgatttgttctttgcga atgaaaagga gggccacttt 420 cacaacatca tcatgccgaa cgtctacattcgtatttttc cttacggttc cgtactatac 480 agcatcagga tatcgcttac tttggcgtgtcctatgaatc tgaaactgta tccgctcgat 540 aggcaggtgt gctctctccg gatggccagttatggttgga ccacaaacga tctggtgttt 600 ttgtggaagg aaggtgaccc ggtgcaggttgtcaagaatc tacatctgcc caggtttacg 660 ttggagaagt tcttgacgga ttattgtaacagcaaaacca ataccggtga atacagttgc 720 ctgaaggtcg acctgctctt taaacgagagttctcgtact acctgatcca gatctacatt 780 ccttgttgca tgttggtgat cgtttcctgggtgtcgttct ggttggacca gggagcggtt 840 ccggccagag tatcactggg tgtgaccactctcctcacca tggccaccca gacgtcgggc 900 ataaacgcct ccctgccgcc agtgtcctacacaaaagcca tcgacgtctg gaccggagtc 960 tgcctcacgt tcgtcttcgg ggctttgctcgaattcgccc tcgtcaacta cgcctccaga 1020 tccgatatgc acagggaaaa catgaagaaaaagcgcaggg aacttgaaca agcagccagc 1080 ctggacgccg cctccgacct gatggacggcactgatggca cttttgctat gaagcctctg 1140 gtacgccact ccgtcgacgc cgtcggtctcgataaggttc gtcagtgcga gatacacatg 1200 cagccggcgt ccaggcagaa ctgctgcaggagctggataa gcaaattccc gacgaggtcg 1260 aaacgcatcg acgtcatatc aagaatcactttcccgctgg tgtttgcttt gttcaatctg 1320 gtgtactggt cgacctattt gttcagggacgaggcggagg agaattag 1368 2 455 PRT ctenocephalides felis 2 Met Asp SerIle Ser Leu Leu Leu Leu Leu Ile Thr Cys Leu Ser Leu 1 5 10 15 His ThrCys Leu Ser Ala Asn Ala Lys Pro Arg Leu Gly Gly Gly Lys 20 25 30 Glu AsnPhe Arg Ala Lys Glu Lys Gln Val Leu Asp Gln Ile Leu Gly 35 40 45 Pro GlyHis Tyr Asp Ala Arg Ile Arg Pro Ser Gly Val Asn Gly Thr 50 55 60 Gly AspGly Pro Thr Val Val Ala Val Asn Ile Tyr Leu Arg Ser Ile 65 70 75 80 SerGlu Ile Asp Asp Tyr Lys Met Glu Tyr Ser Val Gln Leu Thr Phe 85 90 95 ArgGlu Gln Trp Gln Asp Glu Arg Leu Lys Phe Asn Asp Phe Gly Gly 100 105 110Arg Leu Lys Tyr Leu Thr Leu Thr Glu Ala Ser Arg Val Trp Met Pro 115 120125 Asp Leu Phe Phe Ala Asn Glu Lys Glu Gly His Phe His Asn Ile Ile 130135 140 Met Pro Asn Val Tyr Ile Arg Ile Phe Pro Tyr Gly Ser Val Leu Tyr145 150 155 160 Ser Ile Arg Ile Ser Leu Thr Leu Ala Cys Pro Met Asn LeuLys Leu 165 170 175 Tyr Pro Leu Asp Arg Gln Val Cys Ser Leu Arg Met AlaSer Tyr Gly 180 185 190 Trp Thr Thr Asn Asp Leu Val Phe Leu Trp Lys GluGly Asp Pro Val 195 200 205 Gln Val Val Lys Asn Leu His Leu Pro Arg PheThr Leu Glu Lys Phe 210 215 220 Leu Thr Asp Tyr Cys Asn Ser Lys Thr AsnThr Gly Glu Tyr Ser Cys 225 230 235 240 Leu Lys Val Asp Leu Leu Phe LysArg Glu Phe Ser Tyr Tyr Leu Ile 245 250 255 Gln Ile Tyr Ile Pro Cys CysMet Leu Val Ile Val Ser Trp Val Ser 260 265 270 Phe Trp Leu Asp Gln GlyAla Val Pro Ala Arg Val Ser Leu Gly Val 275 280 285 Thr Thr Leu Leu ThrMet Ala Thr Gln Thr Ser Gly Ile Asn Ala Ser 290 295 300 Leu Pro Pro ValSer Tyr Thr Lys Ala Ile Asp Val Trp Thr Gly Val 305 310 315 320 Cys LeuThr Phe Val Phe Gly Ala Leu Leu Glu Phe Ala Leu Val Asn 325 330 335 TyrAla Ser Arg Ser Asp Met His Arg Glu Asn Met Lys Lys Lys Arg 340 345 350Arg Glu Leu Glu Gln Ala Ala Ser Leu Asp Ala Ala Ser Asp Leu Met 355 360365 Asp Gly Thr Asp Gly Thr Phe Ala Met Lys Pro Leu Val Arg His Ser 370375 380 Val Asp Ala Val Gly Leu Asp Lys Val Arg Gln Cys Glu Ile His Met385 390 395 400 Gln Pro Ala Ser Arg Gln Asn Cys Cys Arg Ser Trp Ile SerLys Phe 405 410 415 Pro Thr Arg Ser Lys Arg Ile Asp Val Ile Ser Arg IleThr Phe Pro 420 425 430 Leu Val Phe Ala Leu Phe Asn Leu Val Tyr Trp SerThr Tyr Leu Phe 435 440 445 Arg Asp Glu Ala Glu Glu Asn 450 455 3 1297DNA ctenocephalides felis 3 atggacagca ttagtttgct cctacttttg ataacatgtctaagtctaca cacatgctta 60 tctgcaaatg caaaacctcg tctaggaggc ggcaaagaaaatttcagggc caaagaaaag 120 caagttctgg accaaatttt aggcccaggc cattacgatgccagaataag gccttctgga 180 gtcaatggaa ctggaataca gtgtccagtt aactttcagggaacaatggc aggatgagag 240 gttgaaattt aacgactttg gaggtcgttt aaaatacttaacactaaccg aagcaagtcg 300 tgtatggatg cccgatttgt tctttgcgaa tgaaaaggagggccactttc acaacatcat 360 catgccgaac gtctacattc gtatttttcc ttacggttccgtactataca gcatcaggat 420 atcgcttact ttggcgtgtc ctatgaatct gaaactgtatccgctcgata ggcaggtgtg 480 ctctctccgg atggccagtt atggttggac cacaaacgatctggtgtttt tgtggaagga 540 aggtgacccg gtgcaggttg tcaagaatct acatctgcccaggtttacgt tggagaagtt 600 cttgacggat tattgtaaca gcaaaaccaa taccggtgaatacagttgcc tgaaggtcga 660 cctgctcttt aaacgagagt tctcgtacta cctgatccagatctacattc cttgttgcat 720 gttggtgatc gtttcctggg tgtcgttctg gttggaccagggagcggttc cggccagagt 780 atcactgggt gtgaccactc tcctcaccat ggccacccagacgtcgggca taaacgcctc 840 cctgccgcca gtgtcctaca caaaagccat cgacgtctggaccggagtct gcctcacgtt 900 cgtcttcggg gctttgctcg aattcgccct cgtcaactacgcctccagat ccgatatgca 960 cagggaaaac atgaagaaaa agcgcaggga acttgaacaagcagccagcc tggacgccgc 1020 ctccgacctg atggacggca ctgatggcac ttttgctatgaagcctctgg tacgccactc 1080 cgtcgacgcc gtcggtctcg ataaggttcg tcagtgcgagatacacatgc agccggcgtc 1140 caggcagaac tgctgcagga gctggataag caaattcccgacgaggtcga aacgcatcga 1200 cgtcatatca agaatcactt tcccgctggt gtttgctttgttcaatctgg tgtactggtc 1260 gacctatttg ttcagggacg aggcggagga gaattag 12974 78 PRT ctenocephalides felis 4 Met Asp Ser Ile Ser Leu Leu Leu Leu LeuIle Thr Cys Leu Ser Leu 1 5 10 15 His Thr Cys Leu Ser Ala Asn Ala LysPro Arg Leu Gly Gly Gly Lys 20 25 30 Glu Asn Phe Arg Ala Lys Glu Lys GlnVal Leu Asp Gln Ile Leu Gly 35 40 45 Pro Gly His Tyr Asp Ala Arg Ile ArgPro Ser Gly Val Asn Gly Thr 50 55 60 Gly Ile Gln Cys Pro Val Asn Phe GlnGly Thr Met Ala Gly 65 70 75 5 21 DNA Artificial Sequenceoligonucleotide 5 ctcagagtca ggatccggct a 21 6 21 DNA ArtificialSequence oligonucleotide 6 ctgaaagtta actggacact g 21 7 751 DNActenocephalides felis 7 aactagtgga tcccccgggc tgcaggattc ggcacgagaattttttaaaa taatcctcaa 60 cagcatgata caagaggatg attttatgat ccctgtaaacacttgcttga attttagatt 120 gcaactggag gctccgctga cactctctct tgttcgagcacaggaattgc tcgacatctg 180 gtcaaacgcg ggctacttca taatatccga cgatgacaatttaatgttcg gagcaagaac 240 aattgcagaa tttgaagtgt actttaacga tacattcgaaggacgcatga aaatgtgcac 300 gatgtgcatg ttgcccacct tctattgacc agcaagcaccccttcgccgg tgagcatgtc 360 acccaccgac aggcgccttc tgtgcgccct cgacgacctgcacttagcgg ttgctaagaa 420 gccctaagaa gccgagacgg ttcgcttcgc ccgggggcgattcctcacga tgcacaagcg 480 gaggcgcaag aggctgacga cgaggagcct cagagtcaggatccggctat attggacgat 540 atgctgcatg gtccctgtca tacaaatact ccttcgccttcactggaacc aaccaagact 600 gtccccacgt gtccgacatc agttgaagga aattctgtgacgacatggca acacttttgt 660 tcaggaacaa caataacatc atcgacacag aatatcggcgaagcctattc ttcgattcaa 720 gaagaagaat ttcttcactt tatcttcagg g 751 8 532DNA ctenocephalides felis 8 tcagagtcag gatccggcta tattggacga tatgctgcatggtccctgtc atacaaatac 60 tccttcgcct tcactggaac caaccaagac tgtccccacgtgtccgacat cagttgaagg 120 aaattctgtg acgacatggc aacacttttg ttcaggaacaacaataacat catcgacaca 180 gaatatcggc gaagcctatt cttcgattca agaagaagaatttcttcact ttatcttcag 240 ggatggacag cattagtttg ctcctacttt tgataacatgtctaagtcta cacacatgct 300 tatctgcaaa tgcaaaacct cgtctaggag gcggcaaagaaaatttcagg gccaaagaaa 360 agcaagttct ggaccaaatt ttaggcccag gccattacgatgccagaata aggccttctg 420 gagtcaatgg aactggagac ggtccgaccg tggtagcagtcaacatctat ctgagatcaa 480 tcagcgaaat agatgactac aaaatggaat acagtgtccagttaactttc ag 532

What is claimed:
 1. A purified DNA molecule encoding a C. felis GluClchannel protein wherein said protein comprises the amino acid sequenceas follows: MDSISLLLLL ITCLSLHTCL SANAKPRLGG GKENFRAKEK QVLDQILGPGHYDARIRPSG VNGTGDGPTV VAVNIYLRSI SEIDDYKMEY SVQLTFREQW QDERLKFNDFGGRLKYLTLT EASRVWMPDL FFANEKEGHF HNIIMPNVYI RIFPYGSVLY SIRISLTLACPMNLKLYPLD RQVCSLRMAS YGWTTNDLVF LWKEGDPVQV VKNLHLPRFT LEKFLTDYCNSKTNTGEYSC LKVDLLFKRE FSYYLIQIYI PCCMLVIVSW VSFWLDQGAV PARVSLGVTTLLTMATQTSG INASLPPVSY TKAIDVWTGV CLTFVFGALL EFALVNYASR SDMHRENMKKKRRELEQAAS LDAASDLMDG TDGTFAMKPL VRHSVDAVGL DKVRQCEIHM QPASRQNCCRSWISKFPTRS KRIDVISRIT FPLVFALFNL VYWSTYLFRD EAEEN,

as set forth in three-letter abbreviation in SEQ ID NO:2.
 2. Anexpression vector for expressing a C. felis GluCl channel protein in arecombinant host cell wherein said expression vector comprises a DNAmolecule of claim
 1. 3. A host cell which expresses a recombinant C.felis GluCl channel protein wherein said host cell contains theexpression vector of claim
 2. 4. A process for expressing a C. felisGluCl channel protein in a recombinant host cell, comprising: (a)transfecting the expression vector of claim 2 into a suitable host cell;and, (b) culturing the host cells of step (a) under conditions whichallow expression of said C. felis GluCl channel protein from saidexpression vector.
 5. A purified DNA molecule encoding a C. felis GluClchannel protein wherein said protein consists of the amino acid sequenceas follows: MDSISLLLLL ITCLSLHTCL SANAKPRLGG GKENFRAKEK QVLDQILGPGHYDARIRPSG VNGTGDGPTV VAVNIYLRSI SEIDDYKMEY SVQLTFREQW QDERLKFNDFGGRLKYLTLT EASRVWMPDL FFANEKEGHF HNIIMPNVYI RIFPYGSVLY SIRISLTLACPMNLKLYPLD RQVCSLRMAS YGWTTNDLVF LWKEGDPVQV VKNLHLPRFT LEKFLTDYCNSKTNTGEYSC LKVDLLFKRE FSYYLIQIYI PCCMLVIVSW VSFWLDQGAV PARVSLGVTTLLTMATQTSG INASLPPVSY TKAIDVWTGV CLTFVFGALL EFALVNYASR SDMHRENMKKKRRELEQAAS LDAASDLMDG TDGTFAMKPL VRHSVDAVGL DKVRQCEIHM QPASRQNCCRSWISKFPTRS KRIDVISRIT FPLVFALFNL VYWSTYLFRD EAEEN,

as set forth in three-letter abbreviation in SEQ ID NO:2.
 6. Anexpression vector for expressing a C. felis GluCl channel protein in arecombinant host cell wherein said expression vector comprises a DNAmolecule of claim
 5. 7. A host cell which expresses a recombinant C.felis GluCl channel protein wherein said host cell contains theexpression vector of claim
 6. 8. A process for expressing a C. felisGluCl channel protein in a recombinant host cell, comprising: (a)transfecting the expression vector of claim 6 into a suitable host cell;and, (b) culturing the host cells of step (a) under conditions whichallow expression of said C. felis GluCl channel protein from saidexpression vector.
 9. A purified DNA molecule encoding a recombinant C.felis GluCl channel protein wherein said DNA molecule comprises thenucleotide sequence as set forth in SEQ ID NO:1, as follows: ATGGACAGCATTAGTTTGCT CCTACTTTTG ATAACATGTC TAAGTCTACA CACATGCTTA TCTGCAAATGCAAAACCTCG TCTAGGAGGC GGCAAAGAAA ATTTCAGGGC CAAAGAAAAG CAAGTTCTGGACCAAATTTT AGGCCCAGGC CATTACGATG CCAGAATAAG GCCTTCTGGA GTCAATGGAACTGGAGACGG TCCGACCGTG GTAGCAGTCA ACATCTATCT GAGATCAATC AGCGAAATAGATGACTACAA AATGGAATAC AGTGTCCAGT TAACTTTCAG GGAACAATGG CAGGATGAGAGGTTGAAATT TAACGACTTT GGAGGTCGTT TAAAATACTT AACACTAACC GAAGCAAGTCGTGTATGGAT GCCCGATTTG TTCTTTGCGA ATGAAAAGGA GGGCCACTTT CACAACATCATCATGCCGAA CGTCTACATT CGTATTTTTC CTTACGGTTC CGTACTATAC AGCATCAGGATATCGCTTAC TTTGGCGTGT CCTATGAATC TGAAACTGTA TCCGCTCGAT AGGCAGGTGTGCTCTCTCCG GATGGCCAGT TATGGTTGGA CCACAAACGA TCTGGTGTTT TTGTGGAAGGAAGGTGACCC GGTGCAGGTT GTCAAGAATC TACATCTGCC CAGGTTTACG TTGGAGAAGTTCTTGACGGA TTATTGTAAC AGCAAAACCA ATACCGGTGA ATACAGTTGC CTGAAGGTCGACCTGCTCTT TAAACGAGAG TTCTCGTACT ACCTGATCCA GATCTACATT CCTTGTTGCATGTTGGTGAT CGTTTCCTGG GTGTCGTTCT GGTTGGACCA GGGAGCGGTT CCGGCCAGAGTATCACTGGG TGTGACCACT CTCCTCACCA TGGCCACCCA GACGTCGGGC ATAAACGCCTCCCTGCCGCC AGTGTCCTAC ACAAAAGCCA TCGACGTCTG GACCGGAGTC TGCCTCACGTTCGTCTTCGG GGCTTTGCTC GAATTCGCCC TCGTCAACTA CGCCTCCAGA TCCGATATGCACAGGGAAAA CATGAAGAAA AAGCGCAGGG AACTTGAACA AGCAGCCAGC CTGGACGCCGCCTCCGACCT GATGGACGGC ACTGATGGCA CTTTTGCTAT GAAGCCTCTG GTACGCCACTCCGTCGACGC CGTCGGTCTC GATAAGGTTC GTCAGTGCGA GATACACATG CAGCCGGCGTCCAGGCAGAA CTGCTGCAGG AGCTGGATAA GCAAATTCCC GACGAGGTCG AAACGCATCGACGTCATATC AAGAATCACT TTCCCGCTGG TGTTTGCTTT GTTCAATCTG GTGTACTGGTCGACCTATTT GTTCAGGGAC GAGGCGGAGG AGAATTAG, (SEQ ID NO:1).


10. An expression vector for expressing a recombinant C. felis GluClchannel protein wherein said expression vector comprises a DNA moleculeof claim
 9. 11. A host cell which expresses a recombinant recombinant C.felis GluCl channel protein wherein said host cell contains theexpression vector of claim
 10. 12. A process for expressing arecombinant C. felis GluCl channel protein in a recombinant host cell,comprising: (a) transfecting the expression vector of claim 10 into asuitable host cell; and, (b) culturing the host cells of step (a) underconditions which allow expression of said recombinant C. felis GluClchannel protein from said expression vector.
 13. A purified DNA moleculeencoding a recombinant C. felis GluCl channel protein wherein said DNAmolecule consists of the peptide sequence as set forth in SEQ ED NO:1,as follows: ATGGACAGCA TTAGTTTGCT CCTACTTTTG ATAACATGTC TAAGTCTACACACATGCTTA TCTGCAAATG CAAAACCTCG TCTAGGAGGC GGCAAAGAAA ATTTCAGGGCCAAAGAAAAG CAAGTTCTGG ACCAAATTTT AGGCCCAGGC CATTACGATG CCAGAATAAGGCCTTCTGGA GTCAATGGAA CTGGAGACGG TCCGACCGTG GTAGCAGTCA ACATCTATCTGAGATCAATC AGCGAAATAG ATGACTACAA AATGGAATAC AGTGTCCAGT TAACTTTCAGGGAACAATGG CAGGATGAGA GGTTGAAATT TAACGACTTT GGAGGTCGTT TAAAATACTTAACACTAACC GAAGCAAGTC GTGTATGGAT GCCCGATTTG TTCTTTGCGA ATGAAAAGGAGGGCCACTTT CACAACATCA TCATGCCGAA CGTCTACATT CGTATTTTTC CTTACGGTTCCGTACTATAC AGCATCAGGA TATCGCTTAC TTTGGCGTGT CCTATGAATC TGAAACTGTATCCGCTCGAT AGGCAGGTGT GCTCTCTCCG GATGGCCAGT TATGGTTGGA CCACAAACGATCTGGTGTTT TTGTGGAAGG AAGGTGACCC GGTGCAGGTT GTCAAGAATC TACATCTGCCCAGGTTTACG TTGGAGAAGT TCTTGACGGA TTATTGTAAC AGCAAAACCA ATACCGGTGAATACAGTTGC CTGAAGGTCG ACCTGCTCTT TAAACGAGAG TTCTCGTACT ACCTGATCCAGATCTACATT CCTTGTTGCA TGTTGGTGAT CGTTTCCTGG GTGTCGTTCT GGTTGGACCAGGGAGCGGTT CCGGCCAGAG TATCACTGGG TGTGACCACT CTCCTCACCA TGGCCACCCAGACGTCGGGC. ATAAACGCCT CCCTGCCGCC AGTGTCCTAC ACAAAAGCCA TCGACGTCTGGACCGGAGTC TGCCTCACGT TCGTCTTCGG GGCTTTGCTC GAATTCGCCC TCGTCAACTACGCCTCCAGA TCCGATATGC ACAGGGAAAA CATGAAGAAA AAGCGCAGGG AACTTGAACAAGCAGCCAGC CTGGACGCCG CCTCCGACCT GATGGACGGC ACTGATGGCA CTTTTGCTATGAAGCCTCTG GTACGCCACT CCGTCGACGC CGTCGGTCTC QATAAGGTTC GTCAGTGCGAGATACACATG CAGCCGGCGT CCAGGCAGAA CTGCTGCAGG AGCTGGATAA GCAAATTCCCGACGAGGTCG AAACGCATCG ACGTCATATC AAQAATCACT TTCCCGCTGG TGTTTGCTTTGTTCAATCTG GTGTACTGGT CGACCTATTT GTTCAGGGAC GAGGCGGAGG AGAATTAG, (SEQ IDNO:1).


14. An expression vector for expressing a recombinant C. felis GluClchannel protein wherein said expression vector comprises a DNA moleculeof claim
 13. 15. A host cell which expresses a recombinant recombinantC. felis GluCl channel protein wherein said host cell contains theexpression vector of claim
 14. 16. A process for expressing arecombinant C. felis GluCl channel protein in a recombinant host cell,comprising: (a) transfecting the expression vector of claim 14 into asuitable host cell; and, (b) culturing the host cells of step (a) underconditions which allow expression of said recombinant C. felis GluClchannel protein from said expression vector.
 17. A purified DNA moleculeencoding a truncated portion of aC. felis GluCl channel protein whereinsaid protein consists of the amino acid sequence as follows: MDSISLLLLLITCLSLHTCL SANAKPRLGG GKENFRAKEK QVLDQILGPG HYDARIRPSG VNGTGIQCPVNFQGTMAG,

as set forth in three-letter abbreviation in SEQ ID NO:4.
 18. Anexpression vector for expressing a C. felis GluCl channel protein in arecombinant host cell wherein said expression vector comprises a DNAmolecule of claim
 17. 19. A host cell which expresses a recombinant C.felis GluCl channel protein wherein said host cell contains theexpression vector of claim
 18. 20. A process for expressing a C. felisGluCl channel protein in a recombinant host cell, comprising: (a)transfecting the expression vector of claim 18 into a suitable hostcell; and, (b) culturing the host cells of step (a) under conditionswhich allow expression of said C. felis GluCl channel protein from saidexpression vector.
 21. A purified DNA molecule encoding a recombinant C.felis GluCl channel protein wherein said DNA molecule consists of thenucleotide sequence as set forth in SEQ ID NO:3, as follows: ATGGACAGCATTAGTTTGCT CCTACTTTTG ATAACATGTC TAAGTCTACA CACATGCTTA TCTGCAAATGCAAAACCTCG TCTAGGAGGC GGCAAAGAAA ATTTCAGGGC CAAAGAAAAG CAAGTTCTGGACCAAATTTT AGGCCCAGGC CATTACGATG CCAGAATAAG GCCTTCTGGA GTCAATGGAACTGGAATACA GTGTCCAGTT AACTTTCAGG GAACAATGGC AGGATGAGAG GTTGAAATTTAACGACTTTG GAGGTCGTTT AAAATACTTA ACACTAACCG AAGCAAGTCG TGTATGGATGCCCGATTTGT TCTTTGCGAA TGAAAAGGAG GGCCACTTTC ACAACATCAT CATGCCGAACGTCTACATTC GTATTTTTCC TTACGGTTCC GTACTATACA GCATCAGGAT ATCGCTTACTTTGGCGTGTC CTATGAATCT GAAACTGTAT CCGCTCGATA GGCAGGTGTG CTCTCTCCGGATGGCCAGTT ATGGTTGGAC CACAAACGAT CTGGTGTTTT TGTGGAAGGA AGGTGACCCGGTGCAGGTTG TCAAGAATCT ACATCTGCCC AGGTTTACGT TGGAGAAGTT CTTGACGGATTATTGTAACA GCAAAACCAA TACCGGTGAA TACAGTTGCC TGAAGGTCGA CCTGCTCTTTAAACGAGAGT TCTCGTACTA CCTGATCCAG ATCTACATTC CTTGTTGCAT GTTGGTGATCGTTTCCTGGG TGTCGTTCTG GTTGGACCAG GGAGCGGTTC CGGCCAGAGT ATCACTGGGTGTGACCACTC TCCTCACCAT GGCCACCCAG ACGTCGGGCA TAAACGCCTC CCTGCCGCCAGTGTCCTACA CAAAAGCCAT CGACGTCTGG ACCGGAGTCT GCCTCACGTT CGTCTTCGGGGCTTTGCTCG AATTCGCCCT CGTCAACTAC GCCTCCAGAT CCGATATGCA CAGGGAAAACATGAAGAAAA AGCGCAGGGA ACTTGAACAA GCAGCCAGCC TGGACGCCGC CTCCGACCTGATGGACGGCA CTGATGGCAC TTTTGCTATG AAGCCTCTGG TACGCCACTC CGTCGACGCCGTCGGTCTCG ATAAGGTTCG TCAGTGCGAG ATACACATGC AGCCGGCGTC CAGGCAGAACTGCTGCAGGA GCTGGATAAG CAAATTCCCG ACGAGGTCGA AACGCATCGA CGTCATATCAAGAATCACTT TCCCGCTGGT GTTTGCTTTG TTCAATCTGG TGTACTGGTC GACCTATTTGTTCAGGGACG AGGCGGAGGA GAATTAG, (SEQ ID NO:3).


22. An expression vector for expressing a recombinant C. felis GluClchannel protein wherein said expression vector comprises a DNA moleculeof claim
 21. 23. A host cell which expresses a recombinant C. felisGluCl channel protein wherein said host cell contains the expressionvector of claim
 22. 24. A process for expressing a recombinant C. felisGluCl channel protein in a recombinant host cell, comprising: (a)transfecting the expression vector of claim 22 into a suitable hostcell; and, (b) culturing the host cells of step (a) under conditionswhich allow expression of said recombinant C. felis GluCl channelprotein from said expression vector.